Mitochondrial Profile and Responses to TGF-? Ligands in Keratoconus.
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ABSTRACT:
SUBMITTER: Sarker-Nag A
PROVIDER: S-EPMC5317202 | biostudies-other | 2016 Jul
REPOSITORIES: biostudies-other
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Mitochondrial Profile and Responses to TGF-β Ligands in Keratoconus.
Sarker-Nag Akhee A Hutcheon Audrey E K AE Karamichos Dimitrios D
Current eye research 20151002 7
<h4>Purpose</h4>Keratoconus (KC) is a complex corneal dystrophy with multifactorial etiology. Previous studies have shown evidence of mitochondrial abnormalities in KC; however, the exact cause of these abnormalities remains unknown. The aim of this study was to identify if transforming growth factor-β (TGF-β) isoforms play a role in the regulation of mitochondrial proteins in human KC cells (HKC).<h4>Materials and methods</h4>Human corneal fibroblasts (HCF) and HKC were isolated and cultured fo ...[more]
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Project description:PURPOSE: To analyze tear protein profile variations in patients with keratoconus (KC) and to compare them with those of control subjects. SUBJECTS AND METHODS: Tears from 12 normal subjects and 12 patients with KC were analyzed by two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry (LC-MS). Analysis of the 2-DE gels was performed using Progenesis SameSpots software (Nonlinear Dynamics). Proteins exhibiting high variation in expression levels (P-value <0.05) were identified using matrix-assisted laser desorption/ionization-TOF spectrometry. For LC-MS analysis, a label-free quantification approach was used. Tears were digested with trypsin, subjected to data-independent acquisition (MS(E)) analysis, and identified proteins were relatively quantified using ProteinLynx Global Server software (Waters). RESULTS: The 2-DE and LC-MS analyses revealed a significant decrease in the levels of members of the cystatin family and an increase in lipocalin-1 in KC patients. A 1.43-fold decrease was observed for cystatin-S by 2-DE, and 1.69- and 1.56-fold for cystatin-SN and cystatin-SA by LC-MS, respectively. The increase in lipocalin-1 was observed by both methods with fold changes of 1.26 in the 2-DE approach and 1.31 according to LC-MS. Significant protein upregulation was also observed for Ig-? chain C and Ig J chain proteins by 2-DE. Levels of lipophilin-C, lipophilin-A, and phospholipase A2 were decreased in tears from KC patients according to LC-MS. Serum albumin was found to be increased in KC patients according to LC-MS. CONCLUSION: The results show differences in the tear protein profile of KC and control subjects. These changes are indicative of alterations in tear film stability and in interactions with the corneal surface in KC patients.
| S-EPMC3178250 | biostudies-literature
Project description:PurposeKeratoconus is characterized by the thinning of corneal stroma, resulting in reduced vision. The exact etiology of keratoconus (KC) is still unknown. The involvement of oxidative stress (OS) in this disease has been reported. However, the exact mechanism of OS in keratoconus is still unknown. Thus we planned this study to screen mitochondrial complex I genes for sequence changes in keratoconus patients and controls, as mitochondrial complex I is the chief source of reactive oxygen species (ROS) production.MethodsA total of 20 keratoconus cases and 20 healthy controls without any ocular disorder were enrolled in this study. Mitochondrial complex I genes (ND1, 2, 3, 4, 4L, 5, and 6) were amplified in all patients and controls using 12 pairs of primers by PCR. After sequencing, DNA sequences were analyzed against the mitochondrial reference sequence NC_012920. Haplogroup frequency based Principle Component Analysis (PCA) was constructed to determine whether the gene pool of keratoconus patients is closer to major populations in India.ResultsDNA sequencing revealed a total 84 nucleotide variations in patients and 29 in controls. Of 84 nucleotide changes, 18 variations were non-synonymous and two novel frame-shift mutations were detected in cases. Non-synonymous mtDNA sequence variations may account for increased ROS and decreased ATP production. This ultimately leads to OS; which is a known cause for variety of corneal abnormalities. Haplotype analysis showed that most of the patients were clustered under the haplogroups: T, C4a2a, R2'TJ, M21'Q1a, M12'G2a2a, M8'CZ and M7a2a, which are present as negligible frequency in normal Indian population, whereas only few patients were found to be a part of the other haplogroups like U7 (Indo-European), R2 and R31, whose origin is contentious.ConclusionsMt complex I sequence variations are the main cause of elevated ROS production which leads oxidative stress. This oxidative stress then starts a cascade of events which ultimately can lead to keratoconus. Prompt antioxidant therapy should be initiated in keratoconus patients to minimize ROS related damage.
| S-EPMC3116726 | biostudies-literature
Project description:Keratoconus, historically viewed as a non-inflammatory disease, is an ectatic corneal disorder associated with progressive thinning of the corneal stroma. Recently, a few inflammatory mediators have been reported to be elevated in the tear fluid of keratoconus patients. Consequently, we investigated a wide range of inflammation regulating cytokines in the tears and sera of keratoconus and control subjects. Interleukin (IL)-1?, IL-4, IL-6, IL-10, IL-12, IL-13, IL-17, interferon (IFN)-?, chemokine C-C motif ligand 5 (CCL5) and tumor necrosis factor (TNF)-? were tested in tear samples and sera of keratoconus and control individuals by multiplex immuno-bead assays. Selected cytokines were further tested by standard ELISA on pooled tear samples. All cytokines in the sera were generally low, with no significant changes between keratoconus and control subjects. However, in tear fluids, clear differences were detected between the two groups. These differences include increased IL-6, and decreased IL-12, TNF-?, IFN-?, IL-4, IL-13 and CCL5 in keratoconus compared to control tear fluids. The decreases in IL-12, TNF-? and CCL5 were statistically significant, while the IL-13 decrease was statistically significant in the severe keratoconus group only. IL-17 could not be detected by multiplex immuno-bead assay, but showed an increase in keratoconus by conventional ELISA on a limited number of pooled tear samples. Our findings confirm increased IL-6, but dispute earlier reports of increased TNF-?, and suggest a cytokine imbalance in keratoconus disrupting corneal homeostasis. Moreover, an increase in IL-17 suggests tissue degenerative processes at work, contributing to the thinning and weakening of the corneal connective tissue in keratoconus.
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Project description:Transforming Growth Factor--beta (TGF?) superfamily ligands, including Activins, Growth and Differentiation Factors (GDFs), and Bone Morphogenetic Proteins (BMPs), are excellent targets for protein-based therapeutics because of their pervasiveness in numerous developmental and cellular processes. We developed a strategy termed RASCH (Random Assembly of Segmental Chimera and Heteromer), to engineer chemically-refoldable TGF? superfamily ligands with unique signaling properties. One of these engineered ligands, AB208, created from Activin-?A and BMP-2 sequences, exhibits the refolding characteristics of BMP-2 while possessing Activin-like signaling attributes. Further, we find several additional ligands, AB204, AB211, and AB215, which initiate the intracellular Smad1-mediated signaling pathways more strongly than BMP-2 but show no sensitivity to the natural BMP antagonist Noggin unlike natural BMP-2. In another design, incorporation of a short N-terminal segment from BMP-2 was sufficient to enable chemical refolding of BMP-9, without which was never produced nor refolded. Our studies show that the RASCH strategy enables us to expand the functional repertoire of TGF? superfamily ligands through development of novel chimeric TGF? ligands with diverse biological and clinical values.
| S-EPMC3208551 | biostudies-literature
Project description:PurposeKeratoconus (KC) is a corneal disorder characterized by corneal ectasia, progressive corneal thinning, and conical protrusion. This study aimed to elucidate the mitochondrial gene profile in Chinese patients with KC, analyze the mitochondrial haplogroup and heteroplasmy, and further explore the association between mitochondrial genes and KC.MethodsMitochondrial sequencing was conducted on 100 patients with KC and 100 matched controls. Haplogroup analysis was conducted with logistic regression analysis. The heteroplasmy was analyzed with ANOVA (ANOVA) and Student t test. Sequence kernel association tests (SKATs) were performed to analyze the association between mitochondrial genes and KC. Mtoolbox, Mitoclass.1, and APOGEE were used to estimate the impact of the identified variants in protein-coding genes. PON-mt-tRNA was used to annotate the impact of the variants in tRNA. RNAstructure was used to predict the secondary structures of native and mutated tRNAs.ResultsWe identified 689 variants in patients with KC and 725 variants in controls (with 308 variants shared by both). The mitochondrial haplogroups exhibited no statistically significant differences between the two groups. Based on the heteroplasmy analysis, the number of heteroplasmic variants in the complete mitochondrial genome, RNA coding regions, and noncoding regions were statistically significantly different in the KC cases and controls (p<0.05). The heteroplasmic levels of the m.16180_16182delAA, m.16182insC, and m.14569 G>C variants in the KC cases were statistically significantly higher than those in the controls (p<0.05). The SKAT analysis showed that the COX3 and TRNH genes were statistically significantly associated with KC (p<0.05). Among the nine variants of COX3 included in the SKAT analysis (m.9300G>A, m.9316T>C, m.9327A>G, m.9355A>G, m.9468A>G, m.9612G>A, m.9804G>A, m.9957G>A, and m.9966 G>A), m.9612G>A was predicted to be deleterious by Mtoolbox. The m.9316T>C, m.9327A>G, m.9355A>G, m.9612G>A, m.9804G>A, and m.9957G>A variants were predicted to be damaging by Mitoclass.1. The m.9355A>G and m.9804G>A variants were predicted to be pathogenic by APOGEE. All identified variants located in TRNH (m.12153C>T, m.12178C>T, and m.12192G>A) were predicted to be neutral by the PON-mt-tRNA website.ConclusionsThis study presents the mitochondrial gene profile of Chinese patients with KC and demonstrated that the COX3 and TRNH genes were associated with KC.
| S-EPMC8116249 | biostudies-literature
Project description:Background: Mitochondrial DNA (mtDNA) variants have been implicated in keratoconus (KC). The present study aimed to characterize the mtDNA heteroplasmy profile in KC and explore the association of mitochondrial heteroplasmic levels with KC. Methods: Mitochondrial sequencing of peripheral blood samples and corneal tomography were conducted in 300 KC cases and 300 matched controls. The number of heteroplasmic and homoplasmic variants was calculated across the mitochondrial genome. Spearman's correlation was used to analyze the correlation between the number of heteroplasmic variants and age. The association of mtDNA heteroplasmic level with KC was analyzed by logistic regression analysis. Moreover, the relationship between mitochondrial heteroplasmic levels and clinical parameters was determined by linear regression analysis. Results: The distribution of mtDNA heteroplasmic variants showed the highest number of heteroplasmic variants in the non-coding region, while the COX3 gene exhibited the highest number in protein-coding genes. Comparisons of the number of heteroplasmic and homoplasmic non-synonymous variants in protein-coding genes revealed no significant differences between KC cases and controls (all p > 0.05). In addition, the number of heteroplasmic variants was positively associated with age in all subjects (r = 0.085, p = 0.037). The logistic regression analyses indicated that the heteroplasmic levels of m.16180_16181delAA was associated with KC (p < 0.005). Linear regression analyses demonstrated that the heteroplasmic levels of m.16180_16181delAA and m.302A>C were not correlated with thinnest corneal thickness (TCT), steep keratometry (Ks), and flat keratometry (Kf) (all p > 0.05) in KC cases and controls separately. Conclusion: The current study characterized the mtDNA heteroplasmy profile in KC, and revealed that the heteroplasmic levels of m.16180_16181delAA were associated with KC.
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Project description:ObjectiveTo investigate the correlation between corneal biomechanical properties and topographic parameters using machine learning networks for automatic severity diagnosis and reference benchmark construction.MethodsThis was a retrospective study involving 31 eyes from 31 patients with keratonus. Two clustering approaches were used (i.e., shape-based and feature-based). The shape-based method used a keratoconus benchmark validated for indicating the severity of keratoconus. The feature-based method extracted imperative features for clustering analysis.ResultsThere were strong correlations between the symmetric modes and the keratoconus severity and between the asymmetric modes and the location of the weak centroid. The Pearson product-moment correlation coefficient (PPMC) between the symmetric mode and normality was 0.92 and between the asymmetric mode and the weak centroid value was 0.75.ConclusionThis study confirmed that there is a relationship between the keratoconus signs obtained from topography and the corneal dynamic behaviour captured by the Corvis ST device. Further studies are required to gather more patient data to establish a more extensive database for validation.
| S-EPMC9247384 | biostudies-literature
Project description:PurposeTo investigate the possible association of oxidative stress with keratoconus (KC), we estimated the changes in relative mitochondrial DNA (mtDNA) content.MethodsThe study included 119 patients with KC and 208 controls matched for gender, ethnicity, and systemic disease status. We selected controls who were older than the patients since the mtDNA copy number tends to increase with age. The age mean (standard deviation) was 26.4(7.6) and 54.5(14.4) years for the patients and controls, respectively. The relative mtDNA copy number was estimated with the real-time quantitative PCR (qPCR) method using ND1 as the mtDNA gene and human globulin (HGB; also known as the cytoglobin gene, CYGB) as the reference single-copy nuclear gene.ResultsThe mean relative mtDNA content was significantly higher in patients with KC (1.20±0.45) than in the normal control subjects (1.04±0.36; p = 0.0004). Subjects with high mtDNA content (>1.259, i.e., greater than 75(th) percentile) were at an increased risk of the disease (odds ratio = 2.62, 95% confidence interval = 1.40 to 4.89; p =0.0025).ConclusionsIncreased mtDNA content in patients with KC may indicate mitochondrial respiratory chain defects and thus mitochondrial-abnormality involvement.
| S-EPMC4153421 | biostudies-literature
Project description:Inflammatory factors have been considered to contribute to keratoconus (KC) pathogenesis. This study aims to determine the immune cells subsets and soluble inflammatory factor profile on the ocular surface of KC patients. 32 KC subjects (51 eyes) across different grades of severity and 15 healthy controls (23 eyes) were included in the study. Keratometry and pachymetry measurements were recorded. Ocular surface immune cells (collected by ocular surface wash) immunophenotyped using flow cytometry include leukocytes, neutrophils, macrophages, natural killer (NK) cells, pan-T cells, gamma delta T (γδT) cells and NKT cells. Tear fluid collected using Schirmer's strip was used to measure 50 soluble factors by multiplex ELISA. Proportions of activated neutrophils, NK cells and γδT cells were significantly increased in KC patients. Significantly higher levels of tear fluid IL-1β, IL-6, LIF, IL-17A, TNFα, IFNα/β/γ, EPO, TGFβ1, PDGF-BB, sVCAM, sL-selectin, granzyme-B, perforin, MMP2, sFasL and IgE, along with significantly lower levels of IL-1α and IL-9 were observed in KC patients. Alterations observed in few of the immuno-inflammatory parameters correlated with grades of disease, allergy, eye rubbing and keratometry or pachymetry measurements. The observation implies a distinct immuno-inflammatory component in KC pathogenesis and its potential as an additional therapeutic target in KC management.
| S-EPMC8536707 | biostudies-literature
Project description:PurposeKeratoconus (KC) is a corneal ectasia characterized by structural changes, resulting in progressive thinning and biomechanical weakening that can lead to worsening visual acuity due to irregular astigmatism. Corneal collagen Crosslinking (CXL) and Intracorneal Ring Segment (ICRS) are widely used treatments in KC disease, but the alterations they cause in biomechanical mediators are still poorly understood. The aim of this study was to analyze the tear proteome profile before and after treatments to identify biomarkers altered by surgery.Materials and methodsAn observational, prospective, case-control pilot study was conducted, analyzing tear samples from KC patients by nano-liquid chromatography-mass spectrometry (nLC-MS/MS). Data are available via ProteomeXchange with identifier PXD035655. Patients with KC who underwent ICRS surgery (n = 4), CXL (n = 4), and healthy subjects (Ctrl, n = 4) were included in this study. Clinical parameters were measured and tear samples were collected before and 18 months after surgery. Proteins with ≥2 expression change and p-value < 0.05 between groups and times were selected to study their role in post-operative corneal changes.ResultsThese analyses led to the identification of 447 tear proteins, some of which were dysregulated in KC patients. In comparisons between the two surgical groups and Ctrls, the biological processes that were altered in KC patients at baseline were those that were dysregulated as a consequence of the disease and not of the surgical intervention. Among the biological processes seen to be altered were: immune responses, cytoskeleton components, protein synthesis and metabolic reactions. When comparing the two treatment groups (ICRS and CXL), the process related to cytoskeleton components was the most altered, probably due to corneal thinning which was more pronounced in patients undergoing CXL.ConclusionThe changes observed in tears after 18 months post-operatively could be due to the treatments performed and the pathology. Among the deregulated proteins detected, A-kinase anchor protein 13 (AKAP-13) deserves special attention for its involvement in corneal thinning, and for its strong overexpression in the tears of patients with more active KC and faster disease progression. However, it should be kept in mind that this is a pilot study conducted in a small number of patients.
| S-EPMC9531826 | biostudies-literature