OBJECTIVE

Ahmed glaucoma valve (AGV) implant is an aqueous shunt device used to control intraocular pressure in glaucoma. Implant failure results from impervious encapsulation of the shunt plate causing increased hydraulic resistance and raised intraocular pressure. We hypothesized that deregulation of fibrosis pathway contributes to capsular resistance. We tested this by studying fibrosis related gene expression in failed AGV implants.

METHODS

Differential gene expression was examined in failed AGV capsules and compared to normal control tenon. Following total RNA extraction, 84 key genes in fibrosis pathway were examined by real-time PCR using RT2 Profiler PCR Array. Relative gene expression was calculated using ΔΔCt method. Gene specific TaqMan assays were used to validate select genes with ≥2 fold differential expression in the array expression profile.

RESULTS

We observed differential expression in several genes in the fibrosis pathway. Almost half (39/84) of examined genes showed ≥2 fold differential expression in majority of capsules examined on the array. TaqMan assays for select genes including CCN2 (CTGF), THBS1, SERPINE1, THBS2, COL3A1, MMP3, and IL1A in an increased validation sample set showed significant changes in expression (p value from <0.001 to 0.022) at a high frequency in concurrence with our array results.

CONCLUSIONS

Pathway-focused analyses identified candidate genes with altered expression providing molecular evidence for deregulation of the fibrosis pathway in AGV failure.

To advance the control of airway epithelial cell function and asthma, we investigated the effects of a new curcumin derivative, CNB001, which possesses improved pharmacological properties. Normal human bronchial epithelial (NHBE) cells were stimulated with synthetic double-stranded RNA, Poly(I:C). CNB001 significantly suppressed IL-6, TNF-α, and GM-CSF production by NHBE cells, and did so more effectively than did curcumin or dexamethasone (DEX). CNB001 significantly inhibited the decrease of E-cadherin mRNA expression and increase of vimentin mRNA expression observed in NHBE cells induced by a combination of TGF-β1 and TNF-α, which are markers of airway remodeling. In NHBE cells stimulated by TGF-β1, CNB001 significantly downregulated the level of active serine peptidase inhibitor clade E member (SERPINE) 1, which is also reported to be related to airway remodeling. Whereas DEX alone significantly increased the active SERPINE1 level, the combination of DEX and CNB001 significantly suppressed active SERPINE1. In addition, CNB001 significantly suppressed neutrophil infiltration, IL-6, TNF-α, IL-13 and active SERPINE1 production in bronchoalveolar lavage fluid of the murine asthma model, which was not observed in the case of DEX. In conclusion, the curcumin derivative, CNB001, is a promising candidate to treat asthma associated with neutrophilic airway inflammation and remodeling.

Hepatic toxicity and metabolic disorders are major adverse effects elicited during the pharmacological treatment of the human immunodeficiency virus (HIV) infection. Efavirenz (EFV), the most widely used non-nucleoside reverse transcriptase inhibitor (NNRTI), has been associated with these events, with recent studies implicating it in stress responses involving mitochondrial dysfunction and oxidative stress in human hepatic cells. To expand these findings, we analyzed the influence of EFV on the expression profile of selected stress and toxicity genes in these cells. Significant up-regulation was observed with Cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), which indicated metabolic stress. Several genes directly related to oxidative stress and damage exhibited increased expression, including Methalothionein 2A (MT2A), Heat shock 70kDa protein 6 (HSPA6), Growth differentiation factor 15 (GDF15) and DNA-damage-inducible transcript 3 (DDIT3). In addition, Early growth response protein 1 (EGR1) was enhanced, whereas mRNA levels of the inflammatory genes Chemokine (C-X-C motif) ligand 10 (CXCL10) and Serpin peptidase inhibitor (nexin, plasminogen activator inhibitor type 1), member 1 (SERPINE1) decreased and increased, respectively. This profile of gene expression supports previous data demonstrating altered mitochondrial function and presence of oxidative stress/damage in EFV-treated hepatic cells, and may be of relevance in the search for molecular targets with therapeutic potential to be employed in the prevention, diagnosis and treatment of the hepatic toxicity associated with HIV therapy.

Hepatoma-derived growth factor (HDGF) has been implicated in the growth and metastasis of various types of human cancer, but the role of HDGF expression in prostate cancer or breast cancer has not been documented. To assess the role of HDGF in the proliferation, migration and invasion by prostate and breast cancer cells, HDGF expression in DU145 and MCF7 cells was knocked down using siRNA, and the effect of such knockdown was assessed by MTS and [3H]-thymidine incorporation Transwell assays. Moreover, we identified differentially expressed genes that might mediate the HDGF-induced cellular effects. Our results demonstrate that down-regulation of HDGF expression significantly reduces the proliferation of both DU145 and MCF7 cells. However, down-regulation of HDGF expression in DU145 inhibited cell migration and invasion, but in MCF7 cells it stimulated cell migration and invasion. This differential effect might result from the differential induction of PIK3R1 or SERPINE1 in the two cell lines upon HDGF-siRNA treatment. In conclusion, HDGF may participate in the pathogenesis of prostate and breast cancer by promoting cell growth and it may be a therapeutic target for these cancers.

Occlusive coronary thrombus formation superimposed on an atherosclerotic plaque is the ultimate event leading to myocardial infarction (MI). Therefore, haemostatic proteins may represent important players in the pathogenesis of MI. It was the objective of this study to evaluate, in a comprehensive way, the role of haemostatic gene polymorphisms in predisposition to premature MI. A total of 810 single nucleotide polymorphisms (SNPs) in 37 genes were assessed for association with MI in a large cohort (1,670 males, 210 females) of Italian patients who suffered from an MI event before the age of 45, and an equal number of controls. Thirty-eight SNPs selected from the literature were genotyped using the SNPlex technology, whereas genotypes for the remaining 772 SNPs were extracted from a previous genome-wide association study. Genotypes were analysed by a standard case-control analysis corrected for classical cardiovascular risk factors, and by haplotype analysis. A weighted Genetic Risk Score (GRS) was calculated. Evidence for association with MI after covariate correction was found for 35 SNPs in 12 loci: F5, PROS1, F11, ITGA2, F12, F13A1, SERPINE1, PLAT, VWF, THBD, PROCR, and F9. The weighted GRS was constructed by including the top SNP for each of the 12 associated loci. The GRS distribution was significantly different between cases and controls, and subjects in the highest quintile had a 2.69-fold increased risk for MI compared with those in the lowest quintile. Our results suggest that a GRS, based on the combined effect of several risk alleles in different haemostatic genes, is associated with an increased risk of MI.

Biomarkers are gaining increasing interest to predict risk but also to aid in diagnostics. Tissue-specific biomarkers are of utmost importance to detect diseases of respective organs. As of yet there are no atrium-specific biomarkers for risk stratification of atrial disease, such as atrial fibrillation. Bioinformatics such as mRNA microarrays can help to detect tissue-enriched and possibly tissue-specific expressed genes that can be targets for biomarkers. We describe an approach to identify genes preferably expressed in atrial cardiomyocytes compared with ventricular cardiomyocytes by RNA microarray and confirmed by quantitative real-time polymerase chain reaction. By this approach we identified several atrium-enriched genes but also ventricle-enriched genes. As expected atrial natriuretic peptide (ANP) mRNA showed higher expression in atrial cardiomyocytes while with adrenergic stimulation expression was almost as high in ventricular as in atrial cells. Brain-type natriuretic peptide (BNP), however, was not different between atrial and ventricular cells giving a possible explanation for increased levels of NT-proBNP in atrial fibrillation patients. Interesting identified candidates are serpine1 and ltbp2 as atrium-enriched genes whereas alpha-adrenergic receptor subtype 1b and S100A1 expression was significantly higher in ventricular cells. The identified genes need to be confirmed in human tissue and might ultimately be tested as potential biomarkers for atrial stress. (Neth Heart J 2010;18:610-4.).

BACKGROUND

SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation.

METHODS

SERPINE2 was purified from mouse seminal vesicle secretion using liquid chromatography (LC) and identified by LC/tandem mass spectrometry. The antiserum against the SERPINE2 protein was raised in rabbits. To reveal the uterine and placental expression of SERPINE2, tissues at various stages were collected for real-time PCR quantification, Western blotting, and immunohistochemical staining.

RESULTS

Serpine2 mRNA was the major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation, although Serpine1 mRNA had higher expression levels than Serpine2 mRNA in the placenta. Plat seemed to be the major PA in the mouse uterus and placenta. Antiserum against the SERPINE2 protein specifically recognized two forms of SERPINE2 and an extra 75-kDa protein, which was probably a complex of SERPINE2 with a certain protease, from among thousands of protein components in the tissue extract as demonstrated by Western blotting. In the uterus, SERPINE2 was primarily localized in luminal and glandular epithelial cells but it also was detected in circular and longitudinal smooth muscle cells during the estrous cycle and lactation. It was prominently expressed in decidual stroma cells, the metrial gland, and endometrial epithelium of the pregnant uterus. In the placenta, SERPINE2 was expressed in trophoblasts of the labyrinth and spongiotrophoblasts. However, its expression was remarkably reduced in giant cells which existed in the giant cell-decidual junction zone. In contrast, prominent expression of SERPINE2 seemed to be detected on clusters of glycogen cells near the junction zone. In addition, yolk sac membranes also showed high expression of SERPINE2.

CONCLUSIONS

These findings indicate that SERPINE2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. It may participate in the PA-modulated tissue remodeling process in the mouse placenta and uterus.

Childhood obesity is rapidly increasing in prevalence. We compared circulating membrane-bound tissue factor (FIII, F3) procoagulant activity (TF-PCA) and plasma markers of coagulation, fibrinolysis and endothelial dysfunction in 21 obese (10·1 ± 1·5 years, mean ± standard deviation) and 22 healthy weight children (9·9 ± 1·6 years), classified by Body Mass Index (BMI). TF-PCA and factor VII coagulant activity (FVII:C), plasminogen activator inhibitor (PAI-1, SERPINE1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were higher in obese children. BMI correlated positively with TF-PCA, FVII:C, and PAI-1. Childhood obesity is associated with a procoagulant state and endothelial dysfunction. Studies are needed to assess whether weight reduction reverses these abnormalities.

Therapy targeting the neurovascular unit may provide effective neuroprotection against neonatal hypoxia-ischemia (HI). We hypothesized that the peripheral injection of hypoxia-preconditioned human umbilical vein endothelial cells (HUVECs) following HI protects against neurovascular damage and provides long-term neuroprotection in a postpartum (P) day-7 rat pup model. Compared with normoxic HUVECs, hypoxic HUVECs showed enhanced migration and angiogenesis in vitro and had augmented migration effects into the brain when administered intraperitoneally in vivo after HI. Moreover, 24 and 72 h post-HI, the hypoxic HUVECs group but not the normoxic HUVECs or culture-medium groups had significantly higher preservation of microvessels and neurons, and attenuation of blood-brain barrier damage than the normal-saline group. Compared to control or normal-saline groups, only the hypoxic HUVECs group had no impaired foot steps and showed a significant reduction of brain area loss at P42. Next-generation sequencing showed hypoxia-induced upregulation and downregulation of 209 and 215 genes in HUVECs, respectively. Upstream regulator analysis by ingenuity pathway analysis (IPA) identified hypoxia-inducible factor 1-alpha as the key predicted activated transcription regulator. After hypoxia, 12 genes (ADAMTS1, EFNA1, HIF1A, LOX, MEOX2, SELE, VEGFA, VEGFC, CX3CL1, HMMR, SDC, and SERPINE) associated with migration and/or angiogenesis were regulated in HUVECs. In addition, 6 genes (VEGFA, VEGFC, NTN4, TGFA, SERPINE1, and CX3CL1) involved in the survival of endothelial and neuronal cells were also markedly altered in hypoxic HUVECs. Thus, cell therapy by using hypoxic HUVECs that enhance migration and neurovascular protection may provide an effective therapeutic strategy for treating neonatal asphyxia.

Infectious complications, sepsis, and multiple organ dysfunction syndrome (MODS) remain important causes for morbidity and mortality in patients who survive the initial trauma. Increasing evidence suggests that genetic variants, particularly single nucleotide polymorphisms (SNPs), are critical determinants for interindividual differences in both inflammatory responses and clinical outcome in sepsis patients. Although the effect of SNPs on sepsis and MODS has been studied in many populations and diseases, this review aimed to summarize the current knowledge on the effect of SNPs on infectious complication specifically in trauma patients. A review of available literature was performed in PubMed database. The following genes have been studied in populations of trauma patients: CD14, HMGB1, IFNG, IL1A, IL1B, IL1RN, IL4, IL6, IL8, IL10, IL17F, IL18, MBL2, MASP2, FCN2, TLR1, TLR2, TLR4, TLR9, TNF, LTA, GR, MYLK, NLRP3, PRDX6, RAGE, HSPA1B, HSPA1L, HSP90, SERPINE1, IRAK1, IRAK3, VEGFA, LY96, ANGPT2, LBP, MicroRNA, and mtDNA. In this review, we discuss the genes of the Pattern Recognition Receptors, Signal Transducing Adaptor Proteins, and Inflammatory Cytokines of the innate immune system. A number of genetic variations have so far been studied in cohorts of trauma patients. Studies are often unique and numbers sometimes small. No definitive conclusions can be reached at this time about the influence of specific sequence variations on outcome in trauma patients.

Aberrant gene expression in the gut mucosa might contribute to the initiation and progression of Crohn's disease (CD). RNA sequencing (RNA-seq) provides precise measurements of expression levels of transcripts and their isoforms. The aim of this study was to use RNA-seq to investigate transcriptomic differences and identify significantly differentially expressed transcripts in inflamed and noninflamed intestinal mucosa of CD patients.

RNA-seq was performed on 13 pairs of inflamed and noninflamed intestinal mucosa from 13 CD patients and on sex-matched normal mucosa of 13 healthy controls. Significantly differentially expressed transcripts were validated by immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay.

RNA-seq revealed genome-wide transcriptomic differences between normal mucosa, noninflamed, and inflamed CD mucosa. Among 950 differentially expressed genes, 19 were up- or downregulated (upregulation: ANGPT2, CHN1, CPXM1, CPZ, CXCL1, FCN3, GJC1, HSD11B1, LZTS1, MEOX1, MMP12, PLA1A, SERPINE1, SGIP1, and TRPC4; downregulation: FAM189A1, PDE6A, SLC38A4, and HMGCS2) with statistical significance (p < 0.01 and q < 0.05). Among them, CXCL1 exhibited the highest fold change between groups. Immunohistochemistry for CXCL1 revealed no expression in normal mucosa, slightly increased expression in noninflamed CD mucosa, and highly increased expression in inflamed CD mucosa. Quantitative reverse transcriptase polymerase chain reaction showed that CXCL1 expression was significantly associated with epithelial damage, increased infiltration of polymorphonuclear leukocytes, and submucosal fibrosis. Serum CXCL1 concentration measured by enzyme-linked immunosorbent assay was better correlated with CD activity index (r = 0.660) than with C-reactive protein (r = 0.204).

RNA-seq revealed transcriptomic differences between normal mucosa, noninflamed CD mucosa, and inflamed CD mucosa. Intestinal and serum CXCL1 was substantially increased with CD activity and can be used as a potential biomarker of CD.

A single microRNA (miRNA) has the potential to regulate thousands of genes and thus govern multiple signaling pathways at once. miR-155 is an oncogenic miRNA which regulates many cellular pathways, designating it as a multifaceted regulator of proliferation, chemo-resistance, and apoptosis. While many singular targeted effects of miR-155 have been defined and an oncogenic role has been attributed to miR-155 expression, the global effect of miR-155 on the cellular transcriptomes of an ER(+) breast cancer cell line has yet to be determined. Here we demonstrate that miR-155 expression increases tumorigenesis in vivo and we determine miR-155 mediated transcriptome changes through next generation sequencing analysis. miR-155 expression alters many signaling pathways, with the chief altered pathway being the MAPK signaling cascade and miR-155 induces shortening of target mRNA 3'UTRs and alternative isoform expression of MAPK related genes. In addition there is an observed increase in protein phosphorylation of components of MAPK signaling including ERK1/2 and AP-1 complex members (Fra-1 and c-Fos) as well as elevated gene expression of MAPK regulated genes Zeb1, Snail, Plaur, and SerpinE1.

BACKGROUND

SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs). PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle.

METHODS

Seven patients who underwent a hysterectomy and samples of 120 archived patients' endometrial curettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting was performed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted to localize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2 in various sub-phases of the menstrual cycle.

RESULTS

The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretory phases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in the myometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysis of expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in the endometrium during the secretory phase compared to the proliferative phase.

CONCLUSIONS

The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicating that it may participate in tissue remodeling involved in implantation.

Colorectal cancer (CRC) is a tumor that derives from the rectum or colon, and colon adenocarcinoma (COAD) is the most common type of CRC. The present study was performed to identify genes that serve critical roles in the survival of patients with COAD. RNA-sequencing data of COAD was extracted from The Cancer Genome Atlas database, which included 480 tumor samples and 41 normal samples. Using the limma package, differential expression analysis was performed to identify the differentially expressed genes (DEGs). In addition, the potential functions and pathways for the identified DEGs were analyzed using the clusterProfiler package. After the samples were divided into high and low expression groups, survival analysis for the two groups was performed using the Kaplan-Meier model. Using Cytoscape software, a protein-protein interaction network was generated for the survival-associated genes. A total of 1,519 DEGs, including 568 upregulated genes and 951 downregulated genes, were identified in the COAD samples. Enrichment analysis suggested that the DEGs were implicated in numerous functional terms and pathways. Furthermore, 109 DEGs were identified to be survival-associated genes in COAD. According to the degrees of the network nodes, 5-hydroxytryptamine receptor 1D (HTR1D), TIMP metallopeptidase inhibitor 1 (TIMP1), serpin family E member 1 (SERPINE1), matrix metallopeptidase 3 (MMP3) and cannabinoid receptor 2 (CNR2) were key nodes, and the expression levels of these genes were analyzed in clinical samples of CRC. Therefore, the results of the present study suggest HTR1D, TIMP1, SERPINE1, MMP3 and CNR2 may affect the prognosis of patients with COAD.

Graves' ophthalmopathy (GO), a complication of Graves' disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-β1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-β1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy.

In this study, we aimed to explore the molecular mechanisms of and genetic factors influencing diabetic nephropathy (DN). Gene expression profiles associated with DN were obtained from the GEO database (Accession no. GSE20844). The differentially expressed genes (DEGs) between diabetic mice and non-diabetic mice were screened. Subsequently, the DEGs were subjected to functional and pathway analysis. The protein-protein interaction (PPI) network was constructed and the transcription factors (TFs) were screened among the DEGs. A total of 92 upregulated and 118 downregulated genes were screened. Pathway analysis revealed that the p53 signaling pathway, the transforming growth factor (TGF)-β signaling pathway and the mitogen-activated protein kinase (MAPK) signaling pathway were significantly enriched by upregulated genes. Serpine1 (also known as plasminogen activator inhibitor-1), early growth response 1 (Egr1) and Mdk were found to be significant nodes in the PPI network by three methods. A total of 12 TFs were found to be differentially expressed, of which nuclear receptor subfamily 4, group A, member 1 (Nr4a1) and peroxisome proliferator-activated receptor gamma (Pparg) were found to have multiple interactions with other DEGs. We demonstrated that the p53 signaling pathway, the TGF-β signaling pathway and the MAPK signaling pathway were dysregulated in the diabetic mice. The significant nodes (Serpine1, Egr1 and Mdk) and differentially expressed TFs (Nr4a1 and Pparg) may provide a novel avenue for the targeted therapy of DN.

This study aimed to assess the fibrinolytic inhibitors and their association with thrombosis in Behçet disease. Thrombin activatable fibrinolysis inhibitor (TAFI) (P < 0.001) and plasminogen activator inhibitor-1 (PAI-1) levels (P = 0.022) were significantly higher in 79 patients than in 84 controls. No significant differences were observed in CPB2 (TAFI) Thr325Ile and SERPINE1 (PAI1) 4G/5G polymorphism distribution between patients and controls. TAFI activity levels were significantly higher in patients with thrombosis than in those without thrombosis (P = 0.024). In conclusion, the increased TAFI levels in Behçet disease could contribute to the increased risk of thrombosis observed in these patients.

Tumor overexpression of urokinase-type plasminogen activator (uPA) and its specific inhibitor SerpinE1 (plasminogen activator inhibitor type-1) correlates with poor prognosis and increased metastatic potential. Conversely, tumor expression of uPA and another specific inhibitor, SerpinB2 (plasminogen activator inhibitor type-2), are associated with favorable outcome and relapse-free survival. It is not known how overexpression of these uPA inhibitors results in such disparate outcomes. A possible explanation may be related to the presence of a proposed low density lipoprotein receptor (LDLR)-binding motif in SerpinE1 responsible for mitogenic signaling via ERK that is absent in SerpinB2. We now show that complementation of such a LDLR-binding motif in SerpinB2 by mutagenesis of two key residues enabled high affinity binding to very LDLR (VLDLR). Furthermore, the VLDLR-binding SerpinB2 form behaved in a manner indistinguishable from SerpinE1 in terms of enhanced uPA-SerpinB2 complex endocytosis and subsequent ERK phosphorylation and cell proliferation; that is, the introduction of the LDLR-binding motif to SerpinB2 was necessary and sufficient to allow it to acquire characteristics of SerpinE1 associated with malignancy. In conclusion, this study defines the structural elements underlying the distinct interactions of SerpinE1 versus SerpinB2 with endocytic receptors and how differential VLDLR binding impacts on downstream cellular behavior. This has clear relevance to understanding the paradoxical disease outcomes associated with overexpression of these serpins in cancer.

Accumulation of neurotoxic amyloid peptides (Aβ) in the brain, generated by β-site proteolytic processing of the amyloid precursor protein (APP), is the hallmark pathophysiologic feature of Alzheimer's disease. The plasmin-activating cascade, in which urokinase (uPA) and tissue-type (tPA) plasminogen activators convert plasminogen to the broad-spectrum protease plasmin, appears to serve a protective, Aβ-clearing, role in the central nervous system. Plasmin degrades Aβ and catalyzes α- site APP proteolysis generating nontoxic peptides. Plasmin activation in the brain is negatively regulated by the fast-acting clade E serine protease inhibitor (SERPIN) plasminogen activator inhibitor type-1 (PAI-1; SERPINE1) resulting in Aβ accumulation. PAI-1 and its major physiological inducer TGF-β1, moreover, are both increased in Alzheimer's disease models and implicated in the etiology and progression of human neurodegenerative disorders. Current findings support the hypothesis that targeting of PAI-1 function (by small molecule drugs) and/or gene expression (by histone deacetylase inhibitors) may constitute a clinically-relevant molecular approach to the therapy of neurodegenerative diseases associated with increased PAI-1 levels.

Transforming growth factor-β1 (TGF-β)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN). TGF-β regulates gene expression in MCs by altering key chromatin histone modifications at target gene promoters. However, the role of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification is unclear. Here we show that TGF-β reduces H3K27me3 at the Ctgf, Serpine1, and Ccl2 gene promoters in rat MCs (RMCs) and reciprocally up-regulates the expression of these pro-fibrotic and inflammatory genes. In parallel, TGF-β down-regulates Enhancer of Zeste homolog 2 (Ezh2), an H3K27me3 methyltransferase, and decreases its recruitment at Ctgf and Ccl2 but not Serpine1 promoters. Ezh2 knockdown with siRNAs enhances TGF-β-induced expression of these genes, supporting its repressive function. Mechanistically, Ezh2 down-regulation is mediated by TGF-β-induced microRNA, miR-101b, which targets Ezh2 3'-UTR. TGF-β also up-regulates Jmjd3 and Utx in RMCs, suggesting a key role for these H3K27me3 demethylases in H3K27me3 inhibition. In RMCs, Utx knockdown inhibits hypertrophy, a key event in glomerular dysfunction. The H3K27me3 regulators are similarly altered in human and mouse MCs. High glucose inhibits Ezh2 and increases miR-101b in a TGF-β-dependent manner. Furthermore, in kidneys from rodent models of DN, fibrotic genes, miR-101b, and H3K27me3 demethylases are up-regulated, whereas Ezh2 protein levels as well as enrichment of Ezh2 and H3K27me3 at target genes are decreased, demonstrating in vivo relevance. These results suggest that H3K27me3 inhibition by TGF-β via dysregulation of related histone-modifying enzymes and miRNAs augments pathological genes mediating glomerular mesangial dysfunction and DN.

Increased risk of developing metabolic syndrome (MetS) has been associated with the circadian clock genes. In this study, we assessed whether 29 circadian clock-related genes (including ADCYAP1, ARNTL, ARNTL2, BHLHE40, CLOCK, CRY1, CRY2, CSNK1D, CSNK1E, GSK3B, HCRTR2, KLF10, NFIL3, NPAS2, NR1D1, NR1D2, PER1, PER2, PER3, REV1, RORA, RORB, RORC, SENP3, SERPINE1, TIMELESS, TIPIN, VIP, and VIPR2) are associated with MetS and its individual components independently and/or through complex interactions in a Taiwanese population. We also analyzed the interactions between environmental factors and these genes in influencing MetS and its individual components. A total of 3,000 Taiwanese subjects from the Taiwan Biobank were assessed in this study. Metabolic traits such as waist circumference, triglyceride, high-density lipoprotein cholesterol, systolic and diastolic blood pressure, and fasting glucose were measured. Our data showed a nominal association of MetS with several single nucleotide polymorphisms (SNPs) in five key circadian clock genes including ARNTL, GSK3B, PER3, RORA, and RORB; but none of these SNPs persisted significantly after performing Bonferroni correction. Moreover, we identified the effect of GSK3B rs2199503 on high fasting glucose (P = 0.0002). Additionally, we found interactions among the ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, RORA rs8034880, and RORB rs972902 SNPs influenced MetS (P < 0.001 ~ P = 0.002). Finally, we investigated the influence of interactions between ARNTL rs10832020, GSK3B rs2199503, PER3 rs10746473, and RORB rs972902 with environmental factors such as alcohol consumption, smoking status, and physical activity on MetS and its individual components (P < 0.001 ~ P = 0.002). Our study indicates that circadian clock genes such as ARNTL, GSK3B, PER3, RORA, and RORB genes may contribute to the risk of MetS independently as well as through gene-gene and gene-environment interactions.

OBJECTIVE

Stress of the endoplasmic reticulum and oxidative stress play critical roles in the pathogenesis of Fuchs Endothelial Corneal Dystrophy (FECD). In the normal aging cornea, cellular stress has been associated with a loss in proliferative capacity (premature senescence) of corneal endothelial cells (CECs). The present study used a transgenic Col8a2(Q455K/Q455K) knock-in mouse model of early-onset FECD to identify the endothelial expression profile of specific cellular stress response-related targets, which may be relevant to late-onset FECD.

METHODS

The differential endothelial mRNA levels of cellular stress response-related genes were determined in 12-month-old homozygous Col8a2(Q455K/Q455K) mutant and wild-type mice using customized PCR arrays. Result validation and analysis of additional senescence-related transcripts was performed by real-time PCR. Expression of p53 and p21 was assessed by immunofluorescence. Senescence-associated β-galactosidase (SA-β-Gal) activity was investigated by histochemical labeling. Human FECD samples and normal controls were examined for p21 expression by immunohistochemistry.

RESULTS

PCR-array analysis showed greater than 2-fold and/or significantly altered endothelial regulation of 19 cellular stress response-related transcripts in Col8a2(Q455K/Q455K) mutant mice; real-time PCR documented statistically significant upregulation of senescence-associated targets Cdkn1a (p21), Serpine1 (PAI-1), Tagln (Sm22), Fn1 and Clu (ApoJ). Immunofluorescence revealed increased expression of nuclear p53 and p21 in mutant animals. SA-β-Gal staining detected increased proportions of senescent CECs in mutant mice. Human FECD endothelium exhibited increased levels of nuclear p21 protein.

CONCLUSIONS

Our results identify endothelial Cdkn1a (p21) upregulation in a mouse model of early-onset FECD, confirm overexpression of p21 in late-onset human FECD endothelium, and suggest a role for premature senescence in FECD.