Lack of association between polymorphisms of the DNA base excision repair genes MUTYH and hOGG1 and keratoconus in a Polish subpopulation.

INTRODUCTION: Keratoconus (KC) is a non-inflammatory thinning of the cornea and a leading indication for corneal transplantation. Oxidative stress plays a role in the pathogenesis of this disease. The products of the hOGG1 and MUTYH genes play an important role in the repair of oxidatively modified DNA in the base excision repair pathway. We hypothesized that variability in these genes may change susceptibility to oxidative stress and predispose individuals to the development of KC. We investigated the possible association between the c.977C>G polymorphism of the hOGG1 gene (rs1052133) and the c.972G>C polymorphism of the MUTYH gene (rs3219489) and KC occurrence as well as the modulation of this association by some KC risk factors. MATERIAL AND METHODS: A total of 205 patients with KC and 220 controls were included in this study. The polymorphisms were genotyped with polymerase chain reaction (PCR) restriction fragment length polymorphism and PCR-confronting two-pair primer techniques. Differences in genotype and allele frequency distributions were evaluated using the χ(2) test, and KC risk was estimated with an unconditional multiple logistic regression with and without adjustment for co-occurrence of visual impairment, allergies, sex and family history for KC. RESULTS: We did not find any association between the genotypes and combined genotypes of the c.977C>G polymorphism of the hOGG1 gene and the c.972G>C polymorphism of the MUTYH gene and the occurrence of KC. CONCLUSIONS: Our findings suggest that the c.977C>G-hOGG1 polymorphism and the c.972G>C-MUTYH polymorphism may not be linked with KC occurrence in this Polish subpopulation.

Variation in DNA Base Excision Repair Genes in Fuchs Endothelial Corneal Dystrophy.

BACKGROUND Fuchs endothelial corneal dystrophy (FECD) is a corneal disease characterized by abnormalities in the Descemet membrane and the corneal endothelium. The etiology of this disease is poorly understood. An increased level of oxidative DNA damage reported in FECD corneas suggests a role of DNA base excision repair (BER) genes in its pathogenesis. In this work, we searched for the association between variation of the PARP-1, NEIL1, POLG, and XRCC1 genes and FECD occurrence. MATERIAL AND METHODS This study was conducted on 250 FECD patients and 353 controls using polymerase chain reaction-restriction fragment length polymorphism, high-resolution melting analysis, and the TaqMan® SNP Genotyping Assay. RESULTS We observed that the A/A genotype and the A allele of the c.1196A>G polymorphism of the XRCC1 gene were positively correlated with an increased FECD occurrence, whereas the G allele had the opposite effect. A weak association between the C/G genotype of the g.46438521G>C polymorphism of the NEIL1 gene and an increased incidence of FECD was also detected. Haplotypes of both polymorphisms of the XRCC1 were associated with FECD occurrence. No association of the c.2285T>C, c.-1370T>A and c.580C>T polymorphisms of the PARP-1, POLG and XRCC1 genes, respectively, with FECD occurrence was observed. CONCLUSIONS Our results suggest that the c.1196A>G polymorphism in the XRCC1 gene may be an independent genetic risk factor for FECD.

Polymorphism of the APEX nuclease 1 gene in keratoconus and Fuchs endothelial corneal dystrophy.

Human APEX nuclease 1 (APEX1) plays an important role in the repair of oxidative DNA lesions through base excision repair. It may influence the development of oxidative stress-related diseases. The aim of this study was to determine the relationship between the genotypes of the c.444 T>G (rs1130409) and c.-468 T>G (rs1760944) polymorphisms in the APEX1 gene and the occurrence of two oxidative stress-related eye diseases: keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). The study involved 250 patients with KC, 209 patients with FECD, and 350 control subjects. All of the patients and control subjects underwent a detailed ophthalmic examination. The polymorphisms were genotyped by mismatch polymerase chain reaction restriction fragment length polymorphism (mismatch PCR-RFLP). We observed that the G/T and T/T genotypes of the c.-468 T>G polymorphism were respectively associated with a decreased occurrence of KC (OR 0.54, 95% CI 0.37-0.95; p = 0.030) and an increased occurrence of KC (OR 1.87, 95% CI 1.06-3.32; p = 0.032). None of these polymorphisms showed any association with FECD. Furthermore, no other association was observed, including haplotypes of the two polymorphisms. Our findings suggest that the c.-468 T>G polymorphism of the APEX1 gene may play a role in the pathogenesis of KC.

Expression of RUNX2 and its signaling partners TCF7, FGFR1/2 in cleidocranial dysplasia.

RUNX2 is a member of the PEBP2/CBF transcription factors family controlling the expression of genes whose products are essential for bone formation. Mutations in the RUNX2 gene may be associated with cleidocranial dysplasia (CCD), a rare skeletal disease characterized by stature aberrations, delayed closure of the cranial sutures, hypoplastic or aplastic clavicles, and multiple dental abnormalities. As RUNX2 is involved in many signaling pathways, we hypothesize that CCD may be associated with their changes. We determined the expression of RUNX2 and its signaling partners TCF7, involved in canonical Wnt signaling, and fibroblast growth factor receptors, FGFR1 and FGFR2 in periodontum of CCD patients and control individuals. We did not observe any differences between the level of RUNX2, TCF7 and FGFR1/2 mRNA, determined by real-time PCR, in CDD patients and controls. Therefore, RUNX2 signaling pathways with their partners TCF7 and FGFR1/2 may not be involved in CCD pathogenesis.

Undifferentiated bronchial fibroblasts derived from asthmatic patients display higher elastic modulus than their non-asthmatic counterparts.

During asthma development, differentiation of epithelial cells and fibroblasts towards the contractile phenotype is associated with bronchial wall remodeling and airway constriction. Pathological fibroblast-to-myofibroblast transition (FMT) can be triggered by local inflammation of bronchial walls. Recently, we have demonstrated that human bronchial fibroblasts (HBFs) derived from asthmatic patients display some inherent features which facilitate their FMT in vitro. In spite of intensive research efforts, these properties remain unknown. Importantly, the role of undifferentiated HBFs in the asthmatic process was systematically omitted. Specifically, biomechanical properties of undifferentiated HBFs have not been considered in either FMT or airway remodeling in vivo. Here, we combine atomic force spectroscopy with fluorescence microscopy to compare mechanical properties and actin cytoskeleton architecture of HBFs derived from asthmatic patients and non-asthmatic donors. Our results demonstrate that asthmatic HBFs form thick and aligned 'ventral' stress fibers accompanied by enlarged focal adhesions. The differences in cytoskeleton architecture between asthmatic and non-asthmatic cells correlate with higher elastic modulus of asthmatic HBFs and their increased predilection to TGF-ß-induced FMT. Due to the obvious links between cytoskeleton architecture and mechanical equilibrium, our observations indicate that HBFs derived from asthmatic bronchi can develop considerably higher static tension than non-asthmatic HBFs. This previously unexplored property of asthmatic HBFs may be potentially important for their myofibroblastic differentiation and bronchial wall remodeling during asthma development.

Polymorphism of the DNA base excision repair genes in keratoconus.

Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.-1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease.

Polymorphism of the flap endonuclease 1 gene in keratoconus and Fuchs endothelial corneal dystrophy.

Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.-441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.-441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.-441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy.

Polymorphisms of the apoptosis-related FAS and FAS ligand genes in keratoconus and Fuchs endothelial corneal dystrophy.

Keratoconus (KC) is a non-inflammatory eye disease characterized by progressive corneal thinning and asymmetrical conical protrusion of the cornea. Fuchs endothelial corneal dystrophy (FECD) is a degenerative, slowly progressive disease of the corneal endothelium that is characterized by alteration in corneal endothelial cell morphology and progressive loss of these cells. They are unrelated eye diseases that may ultimately lead to vision loss. Their pathogenesis is largely unknown, although impaired apoptosis has been suggested to be responsible for both diseases. Therefore, we studied the frequency of the c.-671A>G polymorphism of the apoptosis-related FAS gene and the c.-844T>C polymorphism of the FAS ligand (FASLG) gene in patients with FECD (221 individuals) or KC (264) and controls (300). Each polymorphism is located within the putative cis-acting element of the respective promoter. Risk of KC or FECD was estimated with unconditional multiple logistic regression with adjustment for various factors, including age, sex, allergies, and family history. The T/T genotype and the T allele of the c.-844T>C polymorphism were associated with increased occurrence of KC, while the C allele was associated with decreased KC occurrence. The G allele of the c.-671A>G polymorphism was associated with increased occurrence of FECD, while the A allele was associated with decreased FECD occurrence. The C/C-A/A combined genotype was associated with reduced risk of FECD, whereas the T/T-G/A combined genotype increased risk of KC. In conclusion, variability in the expression of the FAS and FASLG genes may be involved in the pathogenesis of KC and FECD.

Role of biochemical factors in the pathogenesis of keratoconus.

Keratoconus (KC) is a corneal disease associated with structural abnormalities in the corneal epithelium, Bowman's layer and stroma and altered concentration of tear components. KC corneas show a different pattern of collagen lamellae than their normal counterparts. Also, a reduction of several collagen types in KC epithelium and stroma was observed. Altered expression and/or activity of lysyl oxidase, a critical enzyme of the biogenesis of connective tissue detected in KC corneas, may weaken covalent bonds between collagen and elastin fibrils, what may lead to biomechanical deterioration of the cornea. Increased activity of matrix metalloproteinases observed in KC may induce the degradation of the extracellular matrix causing damage to the cornea. Oxidative and nitrative stress play an important role in KC pathogenesis and KC corneas are characterized by the disturbed lipid peroxidation and nitric oxide pathways. Malfunctioning of these pathways may lead to accumulation of their toxic by-products inducing several detrimental effects, along with apoptosis of the corneal cells, which may result from the loss of ß-actin or increased levels of cytokines, including interleukin-1 and -6. Change in the expression of genes associated with wound healing, including the nerve growth factor and the visual system homeobox 1, may contribute to increased susceptibility of KC corneas to injury. Consequently, biochemical changes may play an important role in KC pathophysiology and, therefore, can be considered in prevention, diagnosis, prognosis and in the therapy of this disease as well.

Polymorphism of the transferrin gene in eye diseases: keratoconus and Fuchs endothelial corneal dystrophy.

Oxidative stress may play a role in the pathogenesis of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Iron may promote the stress by the Fenton reaction, so its homeostasis should be strictly controlled. Transferrin is essential for iron homeostasis because it transports iron from plasma into cells. The malfunction of transferrin, which may be caused by variation in its gene (TF) variation, may contribute to oxidative stress and change KC and FECD risk. To verify this hypothesis we investigated the association between three polymorphisms of the TF gene, g.3296G>A (rs8177178), g.3481A>G (rs8177179), and c.-2G>A (rs1130459), and KC and FECD occurrence. Genotyping was performed in blood lymphocytes in 216 patients with KC, 130 patients with FECD and 228 controls by PCR-RFLP. We studied also the influence of other risk factors. The A/A genotype and the A allele of the g.3296G>A polymorphism were associated with KC occurrence, while the G allele was negatively correlated with it. We observed a decrease in KC occurrence associated with the A/G genotype of the g.3481A>G polymorphism. We did not find any association between the c.-2G>A polymorphism and KC. No association was found between all three polymorphisms and FECD occurrence.

Polymorphisms of the homologous recombination gene RAD51 in keratoconus and Fuchs endothelial corneal dystrophy.

PURPOSE: We investigated the association between genotypes and haplotypes of the c.-61G>T (rs 1801320) and c.-98G>C (rs 1801321) polymorphisms of the RAD51 gene and the occurrence of keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) in dependence on some environmental factors. METHODS: The polymorphisms were genotyped in peripheral blood lymphocytes of 100 KC and 100 FECD patients as well as 150 controls with PCR-RFLP. RESULTS: The G/T genotype of the c.-61G>T polymorphism was associated with significantly increased frequency occurrence of KC (crude OR 2.99, 95% CI 1.75-5.13). On the other hand, the G/G genotype of this polymorphism was positively correlated with a decreased occurrence of this disease (crude OR 0.52, 95% CI 0.31-0.88). We did not find any correlation between genotypes/alleles of the c.-98G>C polymorphism and the occurrence of KC. We also found that the G/G genotype and G allele of the c.-98G>C polymorphism had a protective effect against FECD (crude OR 0.51, 95% CI 0.28-0.92; crude OR 0.53, 95% CI 0.30-0.92, resp.), while the G/C genotype and the C allele increased FECD occurrence (crude OR 1.85, 95% CI 1.01-3.36; crude OR 1.90, 95% CI 1.09-3.29, resp.). CONCLUSIONS: The c.-61T/T and c.-98G>C polymorphisms of the RAD51 gene may have a role in the KC and FECD pathogenesis and can be considered as markers in these diseases.

Oxidative stress in the pathogenesis of keratoconus and Fuchs endothelial corneal dystrophy.

Due to its localization and function, the cornea is regularly exposed to sunlight and atmospheric oxygen, mainly dioxygen, which produce reactive oxygen species (ROS). Therefore, corneal cells are particularly susceptible to oxidative stress. The accumulation of ROS in the cornea may affect signal transduction, proliferation and may also promote cell death. The cornea has several enzymatic and non-enzymatic antioxidants involved in ROS scavenging, but in certain conditions they may not cope with oxidative stress, leading to diseases of the eye. Keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) are multifactorial diseases of the cornea, in which pathogenesis is not fully understood. However, increased levels of oxidative stress markers detected in these disorders indicate that oxidative stress may play an important role in their development and progression. These markers are: (i) decreased levels of non-enzymatic antioxidants, and (ii) decreased expression of genes encoding antioxidative enzymes, including thioredoxin reductase, peroxiredoxins, superoxide dismutase, glutathione S-transferase, and aldehyde dehydrogenase. Moreover, the FECD endothelium displays higher levels of oxidative DNA damage, especially in mitochondrial DNA (mtDNA), whereas KC cornea shows abnormal levels of some components of oxidative phosphorylation encoded by mtDNA. In this review we present some considerations and results of experiments supporting the thesis on the important role of oxidative stress in KC and FECD pathology.

Apigenin inhibits TGF-ß1 induced fibroblast-to-myofibroblast transition in human lung fibroblast populations.

BACKGROUND: Flavonoids are dietary plant compounds suspected to reduce the incidence of chronic diseases in several regions of the world. Due to anti-allergic and anti-inflammatory activities, apigenin (4',5,7,-trihydroxyflavone) is thought to interfere with crucial events in the pathomechanism of asthma. However, the effect of apigenin on TGF-ß-induced fibroblast-to-myofibroblast transition (FMT) in human lung fibroblast populations, a key event in asthma progression, has not yet been addressed. METHODS: Primary human bronchial fibroblasts (HBFs) propagated from ex vivo bronchial biopsies derived from patients with diagnosed asthma and human embryonic lung IMR-90 fibroblasts were cultured in vitro and treated with TGF-ß1 and apigenin. The myofibroblast fraction in fibroblast populations was evaluated by immunocytochemistry. Expression of α-smooth muscle actin (α-SMA) and tenascin C were assessed at the mRNA and protein level by real-time RT-PCR and immunoblotting, respectively. Additionally, proliferation and viability tests and time lapse-monitoring of movement of individual HBFs and IMR-90 cells were evaluated. RESULTS: We show that apigenin attenuates TGF-ß1-induced FMT in cultures of HBFs, and the magnitude of this attenuation was found to be similar to that observed in the established cell line of lung IMR-90 fibroblasts. Notably, FMT inhibition was observed at low (≈10 µM), non-cytotoxic and non-cytostatic apigenin concentrations and could be correlated with the inhibition of α-SMA and tenascin C expression in HBFs at the mRNA level. CONCLUSIONS: Our data are the first to demonstrate that apigenin inhibits the TGF-ß1-induced expansion of hyper-contractile, α-smooth muscle actin - positive myofibroblasts within populations of HBFs derived from asthmatic patients. They also indicate the possible interference of apigenin with bronchial wall remodeling during the asthmatic process in vivo.

[Oxidative stress in the pathogenesis of Fuchs endothelial corneal dystrophy]. / Stres oksydacyjny jako czynnik patogenezy dystrofii Fuchsa.

Fuchs endothelial corneal dystrophy is a disease which occurs after the fourth decade of life. This disorder is characterized by the formation of excrescences growing from the Descemet membrane, called cormea guttata, and changes in the corneal en- dothelial cell density and morphology. The pathogenesis of Fuchs endothelial corneal dystrophy is not completely known. Auto- somal dominant mode of inheritance observed in some cases of Fuchs endothelial corneal dystrophy suggests possible genetic etiology of the disease. Environmental factors also seem to be associated with Fuchs endothelial corneal dystrophy. A growing number of reports suggest an important role of oxidative stress in this disorder. An increased level of toxic products of reactive oxygen species activity and the decreased expression of antioxidant enzymes, including thioredoxin reductase, metallothione- in 3 and superoxide dismutase 2, were detected in corneas of patients with Fuchs endothelial corneal dystrophy. The imbalance between the production and neutralization of reactive oxygen species may result in oxidative stress exerting a harmful effect on cellular components, leading to molecular and cellular damage. Mitochondria may be a key target of alterationsseen in Fuchs endothelial corneal dystrophy. An increased level of oxidative mitochondrial DNA (mtDNA) damage was detected in corneas of patients with Fuchs endothelial corneal dystrophy. Disturbance in mtDNA may cause loss of integrity of inner mitochondrial membrane potential and activate the inner apoptotic pathway. Consequently, oxidative stress may contribute to the changes in endothelial morphology and apoptosis observed in Fuchs endothelial corneal dystrophy.

[Oxidative stress in the pathogenesis of keratoconus]. / Stres oksydacyjny jako czynnik patogenezy stozka rogówki.

Keratoconus is a non-inflammatory corneal disease, which involves changes of the corneal shape, due to thinning of the corneal stroma. The pathogenesis of this disease has remained unclear, but results of many studies indicate that keratoconus is a multifactorial disease. It is hypothesized, that this disorder is associated with both genetic and environmental factors. An increase in toxic products of lipid peroxidation and nitric oxide pathways, as well as decreased levels of some enzymatic and non-enzymatic antioxidants seen in keratoconus, suggest an important role of oxidative stress in the pathogenesis of this disease. It seems that the interactions of reactive oxygen and nitric species with cellular components including nucleic acids, membrane lipids and proteins, may activate a series of events leading to keratoconus. The excess amount of reactive oxygen and nitric species may induce mitochondrial DNA (mtDNA) damage, the extent of which increases in corneas with keratoconus. This damage may disturb the mitochondrial process of oxidative phosphorylation, resulting in further increase in formation of reactive oxygen and nitric species. Furthermore, some elements of oxidative stress can be involved in the activation of certain proteinases and release of lysosomal enzymes, which may be important for corneal thinning in keratoconus.