Fucoxanthin diminishes oxidative stress damage in human placenta-derived mesenchymal stem cells through the PI3K/Akt/Nrf-2 pathway.

Placenta-derived mesenchymal stem cells (PL-MSCs) have therapeutic potential in various clinical contexts due to their regenerative and immunomodulatory properties. However, with increasing age or extensive in vitro culture, their viability and function are gradually lost, thus restricting their therapeutic application. The primary cause of this deterioration is oxidative injury from free radicals. Therefore, enhancing cell viability and restoring cellular repair mechanisms of PL-MSCs in an oxidative stress environment are crucial in this context. Fucoxanthin, a carotenoid derived from brown seaweed, demonstrates antioxidant activity by increasing the production of antioxidant enzymes and lowering the levels of reactive oxygen species (ROS). This study aimed to determine whether fucoxanthin protects PL-MSCs from hydrogen peroxide (H2O2)-induced oxidative stress. After characterization, PL-MSCs were co-treated with fucoxanthin and H2O2 for 24 h (co-treatment) or pre-treated with fucoxanthin for 24 h followed by H2O2 for 24 h (pre-treatment). The effects of fucoxanthin on cell viability and proliferation were examined using an MTT assay. The expression of antioxidant enzymes, PI3K/Akt/Nrf-2 and intracellular ROS production were investigated in fucoxanthin-treated PL-MSCs compared to the untreated group. The gene expression and involvement of specific pathways in the cytoprotective effect of fucoxanthin were investigated by high-throughput NanoString nCounter analysis. The results demonstrated that co-treatment and pre-treatment with fucoxanthin restored the viability and proliferative capacity of PL-MSCs. Fucoxanthin treatment increased the expression of antioxidant enzymes in PL-MSCs cultured under oxidative stress conditions and decreased intracellular ROS accumulation. Markedly, fucoxanthin treatment could restore PI3K/Akt/Nrf-2 expression in H2O2-treated PL-MSCs. High-throughput analysis revealed up-regulation of genes involved in cell survival pathways, including cell cycle and proliferation, DNA damage repair pathways, and down-regulation of genes in apoptosis and autophagy pathways. This study demonstrated that fucoxanthin protects and rescues PL-MSCs from oxidative stress damage through the PI3K/Akt/Nrf-2 pathway. Our data provide the supporting evidence for the use of fucoxanthin as an antioxidant cytoprotective agent to improve the viability and proliferation capacity of PL-MSCs both in vitro and in vivo required to increase the effectiveness of MSC expansion for therapeutic applications.

Analysis of Wild Type and Variant B Cystatin C Interactome in Retinal Pigment Epithelium Cells Reveals Variant B Interacting Mitochondrial Proteins.

Cystatin C, a secreted cysteine protease inhibitor, is abundantly expressed in retinal pigment epithelium (RPE) cells. A mutation in the protein's leader sequence, corresponding to formation of an alternate variant B protein, has been linked with an increased risk for both age-related macular degeneration (AMD) and Alzheimer's disease (AD). Variant B cystatin C displays intracellular mistrafficking with partial mitochondrial association. We hypothesized that variant B cystatin C interacts with mitochondrial proteins and impacts mitochondrial function. We sought to determine how the interactome of the disease-related variant B cystatin C differs from that of the wild-type (WT) form. For this purpose, we expressed cystatin C Halo-tag fusion constructs in RPE cells to pull down proteins interacting with either the WT or variant B form, followed by identification and quantification by mass spectrometry. We identified a total of 28 interacting proteins, of which 8 were exclusively pulled down by variant B cystatin C. These included 18 kDa translocator protein (TSPO) and cytochrome B5 type B, both of which are localized to the mitochondrial outer membrane. Variant B cystatin C expression also affected RPE mitochondrial function with increased membrane potential and susceptibility to damage-induced ROS production. The findings help us to understand how variant B cystatin C differs functionally from the WT form and provide leads to RPE processes adversely affected by the variant B genotype.

PERK/EIF2AK3 integrates endoplasmic reticulum stress-induced apoptosis, oxidative stress and autophagy responses in immortalised retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) performs essential functions for ensuring retinal homeostasis and is a key site for pathogenic changes leading to age-related macular degeneration (AMD). Compromised proteostasis in RPE results in ER stress and ER stress-dependent antioxidant, apoptosis and autophagic responses. ER stress induces the unfolded protein response (UPR) in which EIF2AK3, encoding the protein kinase RNA-like ER kinase (PERK), acts as a key regulator. Downregulated EIF2AK3 gene expression has recently been identified in AMD using human donor RPE, however the molecular mechanisms that integrate the various ER-mediated cellular pathways underpinning progressive RPE dysfunction in AMD have not been fully characterised. This study investigated the downstream effects of PERK downregulation in response to Brefeldin A (BFA)-induced ER stress in ARPE-19 cells. PERK downregulation resulted in increased ER stress and impaired apoptosis induction, antioxidant responses and autophagic flux. ARPE-19 cells were unable to efficiently induce autophagy following PERK downregulation and PERK presented a role in regulating the rate of autophagy induction. The findings support PERK downregulation as an integrative event facilitating dysregulation of RPE processes critical to cell survival known to contribute to AMD development and highlight PERK as a potential future therapeutic target for AMD.

Human chorion-derived mesenchymal stem cells suppress JAK2/STAT3 signaling and induce apoptosis of cholangiocarcinoma cell lines.

Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the damaged epithelial cells of the biliary tract. Previous studies have reported that the multi-potent mesenchymal stem cells (MSCs) activate a series of tumor signaling pathways by releasing several cytokines to influence tumor cell development. However, the roles and mechanisms of human chorion-derived MSCs (CH-MSCs) in cholangiocarcinoma progression have not been fully addressed. This present study aims to examine the effects of conditioned media derived from CH-MSCs (CH-CM) on CCA cell lines and investigate the respective underlying mechanism of action. For this purpose, MSCs were isolated from chorion tissue, and three cholangiocarcinoma cell lines, namely KKU100, KKU213A, and KKU213B, were used. MTT assay, annexin V/PI analysis, and JC-1 staining were used to assess the effects of CH-CM on proliferation and apoptosis of CCA cells, respectively. Moreover, the effect of CH-CM on caspase-dependent apoptotic pathways was also evaluated. The western blotting assay was also used for measuring the expression of JAK2/STAT3 signaling pathway-associated proteins. The results showed that CH-CM suppressed proliferation and promoted apoptosis of CCA cell lines. CH-CM treatment-induced loss of mitochondrial membrane potential (∆Ψm) in CCA cell lines. The factors presented in the CH-CM also inhibited JAK2/STAT3 signaling, reduced the expression of BCL-2, and increased BAX expression in CCA cells. In conclusion, our study suggests that the CH-CM has a potent anti-cancer effect on cholangiocarcinoma cells and thus provides opportunities for use in alternative cell therapy or in combination with a conventional chemotherapeutic drug to increase the efficiency of CCA treatment.

Epigenetic Age Acceleration Is Not Associated with Age-Related Macular Degeneration.

DNA methylation age (DNAm age) estimation is a powerful biomarker of human ageing. To date, epigenetic clocks have not been evaluated in age-related macular degeneration (AMD). Here, we perform genome-wide DNA methylation analyses in blood of AMD patients with a documented smoking history (14 AMD, 16 Normal), identifying loci of differential methylation (DML) with a relaxed p-value criterion (p ≤ 10-4). We conduct DNAm age analyses using the Horvath-multi tissue, Hannum and Skin & Blood epigenetic clocks in both blood and retinal pigment epithelium (RPE). We perform Ingenuity Pathway Analysis Causal Network Analysis (IPA CNA) on the topmost significantly differentially methylated CpG probes in blood and RPE. Results show poor performance of epigenetic clocks in RPE. Epigenetic age acceleration (EAA) was not observed in AMD. However, we observe positive EAA in blood of smokers, and in smokers with AMD. DML analysis revealed hypomethylation at cg04953735 within RPTOR (p = 6.51 × 10-5; Δß = -11.95%). IPA CNA in the RPE also identified RPTOR as the putative master regulator, predicted to be inhibited in AMD. In conclusion, this is the first study evaluating an association of epigenetic ageing in AMD. We posit a role for RPTOR as a common master regulator of methylation changes in the RPE in AMD.

Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

An integrative analysis of the age-associated multi-omic landscape across cancers.

Age is the most important risk factor for cancer, as cancer incidence and mortality increase with age. However, how molecular alterations in tumours differ among patients of different age remains largely unexplored. Here, using data from The Cancer Genome Atlas, we comprehensively characterise genomic, transcriptomic and epigenetic alterations in relation to patients' age across cancer types. We show that tumours from older patients present an overall increase in genomic instability, somatic copy-number alterations (SCNAs) and somatic mutations. Age-associated SCNAs and mutations are identified in several cancer-driver genes across different cancer types. The largest age-related genomic differences are found in gliomas and endometrial cancer. We identify age-related global transcriptomic changes and demonstrate that these genes are in part regulated by age-associated DNA methylation changes. This study provides a comprehensive, multi-omics view of age-associated alterations in cancer and underscores age as an important factor to consider in cancer research and clinical practice.

PERP-ing into diverse mechanisms of cancer pathogenesis: Regulation and role of the p53/p63 effector PERP.

The tetraspan plasma membrane protein PERP (p53 apoptosis effector related to PMP22) is a lesser-known transcriptional target of p53 and p63. A member of the PMP22/GAS3/EMP membrane protein family, PERP was originally identified as a p53 target specifically trans-activated during apoptosis, but not during cell-cycle arrest. Several studies have since shown downregulation of PERP expression in numerous cancers, suggesting that PERP is a tumour suppressor protein. This review focusses on the important advances made in elucidating the mechanisms regulating PERP expression and its function as a tumour suppressor in diverse human cancers, including breast cancer and squamous cell carcinoma. Investigating PERP's role in clinically-aggressive uveal melanoma has revealed that PERP engages a positive-feedback loop with p53 to regulate its own expression, and that p63 is required beside p53 to achieve pro-apoptotic levels of PERP in this cancer. Furthermore, the recent discovery of the apoptosis-mediating interaction of PERP with SERCA2b at the plasma membrane-endoplasmic reticulum interface demonstrates a novel mechanism of PERP stabilisation, and how PERP can mediate Ca2+ signalling to facilitate apoptosis. The multi-faceted role of PERP in cancer, involving well-documented functions in mediating apoptosis and cell-cell adhesion is discussed, alongside PERP's emerging roles in epithelial-mesenchymal transition, and PERP crosstalk with inflammation signalling pathways, and other signalling pathways. The potential for restoring PERP expression as a means of cancer therapy is also considered.

Secretory proteostasis of the retinal pigmented epithelium: Impairment links to age-related macular degeneration.

Secretory proteostasis integrates protein synthesis, processing, folding and trafficking pathways that are essential for efficient cellular secretion. For the retinal pigment epithelium (RPE), secretory proteostasis is of vital importance for the maintenance of the structural and functional integrity of apical (photoreceptors) and basal (Bruch's membrane/choroidal blood supply) sides of the environment it resides in. This integrity is achieved through functions governed by RPE secreted proteins, which include extracellular matrix modelling/remodelling, angiogenesis and immune response modulation. Impaired RPE secretory proteostasis affects not only the extracellular environment, but leads to intracellular protein aggregation and ER-stress with subsequent cell death. Ample recent evidence implicates dysregulated proteostasis as a key factor in the development of age-related macular degeneration (AMD), the leading cause of blindness in the developed world, and research aiming to characterise the roles of various proteins implicated in AMD-associated dysregulated proteostasis unveiled unexpected facets of the mechanisms involved in degenerative pathogenesis. This review analyses cellular processes unveiled by the study of the top 200 transcripts most abundantly expressed by the RPE/choroid in the light of the specialised secretory nature of the RPE. Functional roles of these proteins and the mechanisms of their impaired secretion, due to age and genetic-related causes, are analysed in relation to AMD development. Understanding the importance of RPE secretory proteostasis in relation to maintaining retinal health and how it becomes impaired in disease is of paramount importance for the development and assessment of future therapeutic advancements involving gene and cell therapies.

Increased Rate of Retinal Pigment Epithelial Cell Migration and Pro-Angiogenic Potential Ensuing From Reduced Cystatin C Expression.

Purpose: Variant B precursor cysteine protease inhibitor cystatin C, a known recessive risk factor for developing exudative age-related macular degeneration (AMD), presents altered intracellular trafficking and reduced secretion from retinal pigment epithelial (RPE) cells. Because cystatin C inhibits multiple extracellular matrix (ECM)-degrading cathepsins, this study evaluated the role of this mutation in inducing ECM-related functional changes in RPE cellular behavior. Methods: Induced pluripotent stem cells gene-edited bi-allelically by CRISPR/Cas9 to express the AMD-linked cystatin C variant were differentiated to RPE cells and assayed for their ability to degrade fluorescently labeled ECM proteins. Cellular migration and adhesion on multiple ECM proteins, differences in transepithelial resistance and polarized protein secretion were tested. Vessel formation induced by gene edited cells-conditioned media was quantified using primary human dermal microvascular epithelial cells. Results: Variant B cystatin C-expressing induced pluripotent stem cells-derived RPE cells displayed a significantly higher rate of laminin and fibronectin degradation 3 days after seeding on fluorescently labeled ECM (P < 0.05). Migration on matrigel, collagen IV and fibronectin was significantly faster for edited cells compared with wild-type (WT) cells. Both edited and WT cells displayed polarized secretion of cystatin C, but transepithelial resistance was lower in gene-edited cells after 6 weeks culture, with significantly lower expression of tight junction protein claudin-3. Media conditioned by gene-edited cells stimulated formation of significantly longer microvascular tubes (P < 0.05) compared with WT-conditioned media. Conclusions: Reduced levels of cystatin C lead to changes in the RPE ability to degrade, adhere, and migrate supporting increased invasiveness and angiogenesis relevant for AMD pathology.

Curcumin diethyl disuccinate, a prodrug of curcumin, enhances anti-proliferative effect of curcumin against HepG2 cells via apoptosis induction.

Curcumin (Cur) has been reported to have anti-hepatocellular carcinoma activity but its poor oral bioavailability limits its further development as a chemotherapeutic agent. We synthesized previously a succinate ester prodrug of Cur, curcumin diethyl disuccinate (CurDD) with better chemical stability in a buffer solution pH 7.4. Here, we further investigated and compared the cellular transport and anti-proliferative activity against HepG2 cells of CurDD and Cur. Transport of CurDD across the Caco-2 monolayers provided a significantly higher amount of the bioavailable fraction (BF) of Cur with better cytotoxicity against HepG2 cells compared to that of Cur (p < 0.05). Flow cytometric analysis showed that the BF of CurDD shifted the cell fate to early and late apoptosis to a higher extent than that of Cur. The Western blot analysis revealed that CurDD increased Bax protein expression, downregulated Bcl-2 protein, activated caspase-3 and -9 and increased LC3-II protein level in HepG2 cells. Flow cytometric and immunoblotting results suggest that CurDD can induce HepG2 cell death via an apoptotic pathway. We suggest that CurDD can overcome the limitations of Cur in terms of cellular transport with a potential for further extensive in vitro and in vivo studies of anti-hepatocellular carcinoma effects.

Whole-genome methylation profiling of the retinal pigment epithelium of individuals with age-related macular degeneration reveals differential methylation of the SKI, GTF2H4, and TNXB genes.

BACKGROUND: Age-related macular degeneration (AMD) is a degenerative disorder of the central retina and the foremost cause of blindness. The retinal pigment epithelium (RPE) is a primary site of disease pathogenesis. The genetic basis of AMD is relatively well understood; however, this knowledge is yet to yield a treatment for the most prevalent non-neovascular disease forms. Therefore, tissue-specific epigenetic mechanisms of gene regulation are of considerable interest in AMD. We aimed to identify differentially methylated genes associated with AMD in the RPE and differentiate local DNA methylation aberrations from global DNA methylation changes, as local DNA methylation changes may be more amenable to therapeutic manipulation. METHODS: Epigenome-wide association study and targeted gene expression profiling were carried out in RPE cells from eyes of human donors. We performed genome-wide DNA methylation profiling (Illumina 450k BeadChip array) on RPE cells from 44 human donor eyes (25 AMD and 19 normal controls). We validated the findings using bisulfite pyrosequencing in 55 RPE samples (30 AMD and 25 normal controls) including technical (n = 38) and independent replicate samples (n = 17). Long interspersed nucleotide element 1 (LINE-1) analysis was then applied to assess global DNA methylation changes in the RPE. RT-qPCR on independent donor RPE samples was performed to assess gene expression changes. RESULTS: Genome-wide DNA methylation profiling identified differential methylation of multiple loci including the SKI proto-oncogene (SKI) (p = 1.18 × 10-9), general transcription factor IIH subunit H4 (GTF2H4) (p = 7.03 × 10-7), and Tenascin X (TNXB) (p = 6.30 × 10-6) genes in AMD. Bisulfite pyrosequencing validated the differentially methylated locus cg18934822 in SKI, and cg22508626 within GTF2H4, and excluded global DNA methylation changes in the RPE in AMD. We further demonstrated the differential expression of SKI, GTF2H4, and TNXB in the RPE of independent AMD donors. CONCLUSIONS: We report the largest genome-wide methylation analysis of RPE in AMD along with associated gene expression changes to date, for the first-time reaching genome-wide significance, and identified novel targets for functional and future therapeutic intervention studies. The novel differentially methylated genes SKI and GTF2H4 have not been previously associated with AMD, and regulate disease pathways implicated in AMD, including TGF beta signaling (SKI) and transcription-dependent DNA repair mechanisms (GTF2H4).

CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection.

CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.

Advanced glycation end products-related modulation of cathepsin L and NF-κB signalling effectors in retinal pigment epithelium lead to augmented response to TNFα.

The retinal pigment epithelium (RPE) plays a central role in neuroretinal homoeostasis throughout life. Altered proteolysis and inflammatory processes involving RPE contribute to the pathophysiology of age-related macular degeneration (AMD), but the link between these remains elusive. We report for the first time the effect of advanced glycation end products (AGE)-known to accumulate on the ageing RPE's underlying Bruch's membrane in situ-on both key lysosomal cathepsins and NF-κB signalling in RPE. Cathepsin L activity and NF-κB effector levels decreased significantly following 2-week AGE exposure. Chemical cathepsin L inhibition also decreased total p65 protein levels, indicating that AGE-related change of NF-κB effectors in RPE cells may be modulated by cathepsin L. However, upon TNFα stimulation, AGE-exposed cells had significantly higher ratio of phospho-p65(Ser536)/total p65 compared to non-AGEd controls, with an even higher fold increase than in the presence of cathepsin L inhibition alone. Increased proportion of active p65 indicates an AGE-related activation of NF-κB signalling in a higher proportion of cells and/or an enhanced response to TNFα. Thus, NF-κB signalling modulation in the AGEd environment, partially regulated via cathepsin L, is employed by RPE cells as a protective (para-inflammatory) mechanism but renders them more responsive to pro-inflammatory stimuli.

p63 is required beside p53 for PERP-mediated apoptosis in uveal melanoma.

BACKGROUND: PERP (p53 apoptosis effector related to PMP-22), a transcriptional target of p53, is downregulated and contributes to the impairment of apoptosis in uveal melanoma (UM). Intriguingly, PERP is not induced in UM despite functional p53. p63, located on chromosome 3, which is characteristically altered in high-risk UM, can transactivate PERP. Here, we determine the functional role of p63 expression in the initiation of p53/PERP-mediated apoptosis in UM. METHODS: PERP expression was monitored by quantitative PCR (qPCR) and immunoblotting in UM cell lines treated with DNA-damaging agents. The functional role of p63 was assessed by transient expression of p63-turbo GFP (p63-tGFP) in the apoptosis- resistant, 3q-deficient OCM-1 cells. Expression and localisation of p63, PERP and p53, and induction of apoptosis were characterised by qPCR, immunoblotting and live cell confocal microscopy. RESULTS: PERP expression was significantly downregulated in all UM cell lines. DNA-damaging treatments failed to induce apoptosis and activate PERP in OCM-1 cells, which displayed non-functional levels of p63. Expression of p63-tGFP induced apoptosis with marked increase in PERP expression and associated p53 accumulation. CONCLUSIONS: Lack of p63 contributes to reduced PERP levels and impaired p53-mediated apoptosis in UM. p63 expression is required for PERP-mediated apoptosis in UM.

Amyloid properties of the leader peptide of variant B cystatin C: implications for Alzheimer and macular degeneration.

Variant B (VB) of cystatin C has a mutation in its signal peptide (A25T), which interferes with its processing leading to reduced secretion and partial retention in the vicinity of the mitochondria. There are genetic evidences of the association of VB with Alzheimer's disease (AD) and age-related macular degeneration (AMD). Here, we investigated aggregation and amyloid propensities of unprocessed VB combining computational and in vitro studies. Aggregation predictors revealed the presence of four aggregation-prone regions, with a strong one at the level of the signal peptide, which indeed formed toxic aggregates and mature amyloid fibrils in solution. In light of these results, we propose for the first time the role of the signal peptide in pathogenesis of AD and AMD.

A missense variant in CST3 exerts a recessive effect on susceptibility to age-related macular degeneration resembling its association with Alzheimer's disease.

Age-related macular degeneration (AMD) and Alzheimer's disease (AD) are degenerative, multifactorial diseases involving age-related accumulation of extracellular deposits linked to dysregulation of protein homeostasis. Here, we strengthen the evidence that an nsSNP (p.Ala25Thr) in the cysteine proteinase inhibitor cystatin C gene CST3, previously confirmed by meta-analysis to be associated with AD, is associated with exudative AMD. To our knowledge, this is the first report highlighting a genetic variant that increases the risk of developing both AD and AMD. Furthermore, we demonstrate that the risk associated with the mutant allele follows a recessive model for both diseases. We perform an AMD-CST3 case-control study genotyping 350 exudative AMD Caucasian individuals. Bringing together our data with the previously reported AMD-CST3 association study, the evidence of a recessive effect on AMD risk is strengthened (OR = 1.89, P = 0.005). This effect closely resembles the AD-CST3 recessive effect (OR = 1.73, P = 0.005) previously established by meta-analysis. This resemblance is substantiated by the high correlation between CST3 genotype and effect size across the two diseases (R(2) = 0.978). A recessive effect is in line with the known function of cystatin C, a potent enzyme inhibitor. Its potency means that, in heterozygous individuals, a single functional allele is sufficient to maintain its inhibitory function; only homozygous individuals will lack this form of proteolytic regulation. Our findings support the hypothesis that recessively acting variants account for some of the missing heritability of multifactorial diseases. Replacement therapy represents a translational opportunity for individuals homozygous for the mutant allele.

Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions.

Recently, thrombospondin-1 (TSP-1) has been reported to be critical for maintaining a healthy ocular surface. The purpose of the study was to characterize the expression of TSP-1 and of its receptors CD36 and CD47 in corneal and conjunctival epithelial cells and determine the effect of exogenous TSP-1 treatment on these cells, following the induction of inflammation- and apoptosis-related changes. The expression of TSP-1, CD36 and CD47 by corneal and conjunctival cell lines was firstly characterized by ELISA, immunofluorescence analysis, Western blotting and reverse transcription polymerase chain reaction (RT-PCR). Benzalkonium chloride (BAC) exposure for 5 or 15 min was used as pro-inflammatory and pro-apoptotic stimulus for corneal or conjunctival epithelial cells, respectively. To analyze inflammation and apoptosis-related changes, IL-6 and TGF-ß2 secretion determined by ELISA was used as inflammatory markers, while activated caspase-3/7 levels and cell viability, determined by CellEvent™ Caspase-3/7 Green Detection Reagent and XTT cytotoxicity assay, respectively, were used as apoptotic markers. Changes in CD36 and CD47 mRNA expression were quantified by real time RT-PCR. Corneal epithelial cells secreted and expressed higher protein levels of TSP-1 than conjunctival epithelial cells, although TSP-1 mRNA expression levels were similar and had lower CD36 and CD47, both at protein and mRNA levels. Both cell lines responded to exogenous TSP-1 treatment increasing CD36 at protein and mRNA levels. Blocking experiments revealed a predominance of TSP-1/CD47 rather than TSP-1/CD36 interactions to up-regulate CD36 levels in conjunctival epithelial cells, but not in corneal epithelial cells. BAC exposure increased IL-6 secretion and caspase-3/7 levels and decreased cell viability in both, corneal and conjunctival epithelial cells. Moreover, BAC exposure increased latent TGF-ß2 levels in conjunctival epithelial cells. Interestingly, CD36 mRNA expression was down-regulated after BAC exposure in both cell lines. Exogenous TSP-1 treatment reduced TGF-ß2 up-regulated levels by BAC exposure in conjunctival epithelial cells and less pronounced reduced IL-6 in BAC-exposed corneal epithelial cells. The effect on CD36 and CD47 regulation was less pronounced or even opposite depending on the inflammation- and apoptosis-related markers tested. Our results show evidence of the capacity of corneal and conjunctival epithelial cells to respond to TSP-1 via CD36 or CD47. Experimental simulation of inflammation- and apoptosis-related conditions changed the effects differentially elicited by TSP-1 on corneal and conjunctival epithelial cells, suggesting an unexpected and relevant contribution of TSP-1 on ocular surface homeostasis regulation.

PERP, a host tetraspanning membrane protein, is required for Salmonella-induced inflammation.

Salmonella enterica Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from Salmonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast-two-hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP-22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface.

Age-related changes of cystatin C expression and polarized secretion by retinal pigment epithelium: potential age-related macular degeneration links.

PURPOSE: Cystatin C, a potent cysteine proteinase inhibitor, is abundantly secreted by the RPE and may contribute to regulating protein turnover in the Bruch's membrane (BrM). A cystatin C variant associated with increased risk of developing AMD and Alzheimer's disease (AD) presents reduced secretion levels from RPE. The purpose of this study was to analyze the effects of age and the accumulation of advanced glycation end-products (AGEs) on the expression and secretion of cystatin C by the RPE. METHODS: Confluent monolayers of human fetal RPE (hfRPE) cells were cultured using an in vitro model mimicking extracellular AGE accumulation. Cystatin C expression, secretion, and its polarity were analyzed following culture on AGE-containing BrM mimics (AGEd versus non-AGEd). Monolayer barrier properties were assessed by transepithelial resistance measurements. The relative level of cystatin C protein expression in human RPE in situ was assessed immunohistochemically in relation to age. RESULTS: Advanced glycation end product-exposed RPE monolayers presented significantly decreased cystatin C expression and secretion. Basolateral secretion was fully established by week 8 in non-AGEd conditions. In AGEd cultures, polarity of secretion was impaired despite maintenance of physiological barrier properties of the monolayer. In the macula region of RPE/choroid segments from human eyes, the level of cystatin C protein was reduced with increasing donor age. CONCLUSIONS: Exposure to AGEs reduces expression of cystatin C and affects its normal secretion in cultured RPE. Age-related changes of cystatin C in the RPE from the posterior pole may compromise its extracellular functions, potentially contributing to AMD pathogenesis.