Blood biomarkers for assessment of mitochondrial dysfunction: An expert review.

Although mitochondrial dysfunction is the known cause of primary mitochondrial disease, mitochondrial dysfunction is often difficult to measure and prove, especially when biopsies of affected tissue are not available. In order to identify blood biomarkers of mitochondrial dysfunction, we reviewed studies that measured blood biomarkers in genetically, clinically or biochemically confirmed primary mitochondrial disease patients. In this way, we were certain that there was an underlying mitochondrial dysfunction which could validate the biomarker. We found biomarkers of three classes: 1) functional markers measured in blood cells, 2) biochemical markers of serum/plasma and 3) DNA markers. While none of the reviewed single biomarkers may perfectly reveal all underlying mitochondrial dysfunction, combining biomarkers that cover different aspects of mitochondrial impairment probably is a good strategy. This biomarker panel may assist in the diagnosis of primary mitochondrial disease patients. As mitochondrial dysfunction may also play a significant role in the pathophysiology of multifactorial disorders such as Alzheimer's disease and glaucoma, the panel may serve to assess mitochondrial dysfunction in complex multifactorial diseases as well and enable selection of patients who could benefit from therapies targeting mitochondria.

Increased ratios of complement factors C3a to C3 in aqueous humor and serum mark glaucoma progression.

INTRODUCTION: We recently performed a combined analysis of publicly available proteomic studies of aqueous humor (AH) of patients with primary open angle glaucoma (POAG). This analysis revealed changes in complement protein concentrations in the AH of progressive POAG patients, which suggested that the complement system may play a role in POAG progression. As the proteomic studies could not provide information on the activity of the complement system, we addressed this question in the current study. METHODS: Blood serum and AH were obtained from 30 patients: 10 progressive POAG, 10 stable POAG and, as controls, 10 cataract patients. Glaucoma patients with a visual field Mean Deviation (MD) change of at least 1.0 dB/year were considered progressive; a MD change of less than 0.5 dB/year was considered stable. The ratio between the levels of complement factors C3a and C3 was used as indicator for activation of the complement cascade. The factors were measured with commercially available ELISA kits. RESULTS: AH levels of complement factors C3 and C3a did not significantly differ between groups. In serum, complement factor C3 did not differ between groups whereas C3a was significantly elevated in progressive POAG patients compared to controls (p < 0.05). The resulting complement C3a/C3 ratio was significantly higher in progressive POAG patients in both AH (p < 0.05) and serum (p < 0.01), and this ratio significantly correlated between the two body fluids (p < 0.001). Furthermore, there was a strong correlation between disease progression and C3a/C3 activation ratio both in AH (p < 0.01) and in serum (p < 0.001). The higher the complement C3a/C3 ratio, the faster the disease progression. CONCLUSION: Significant increases in AH and serum complement C3a/C3 ratios were observed in progressive POAG patients but not in stable POAG patients. Furthermore, the complement C3a/C3 ratio correlated strongly with the rate of disease progression in both AH and serum. These findings suggest that activation of the complement system plays a role in glaucoma progression and that progressive glaucoma patients may have systemic changes in complement activation.

Aqueous humor proteome of primary open angle glaucoma: A combined dataset of mass spectrometry studies.

Analysis of the proteins of the aqueous humor can help to elucidate the complex pathogenesis of primary open angle glaucoma. Thanks to advances in liquid chromatography tandem mass spectrometry (LC-MS/MS) it is now possible to identify hundreds of proteins in individual aqueous humor samples without the need to pool samples. We performed a systematic literature search to find publications that performed LC-MS/MS on aqueous humor samples of glaucoma patients and of non-glaucomatous controls. Of the seven publications that we found, we obtained the raw data of three publications. These three studies used glaucoma patients that were clinically similar (i.e. undergoing glaucoma filtration surgery) which prompted us to reanalyse and combine their data. Raw data of each study were analysed separately with the latest version of MaxQuant (version v1.6.11.0). Outcome files were exported to Microsoft Excel. Samples belonging to the same patient were averaged to obtain peptide expression values per individual. We compared the overlap of identified proteins using the VLOOKUP function of Excel and a publicly available Venn diagram software. For the peptide sequences that can belong to multiple proteins (usually of the same protein family), we initially included all possibly identified proteins. This ensured that we would not miss a potential overlap between the studies due to differences in identified peptide counts. Next, of those peptides of which we compared multiple proteins, only one unique protein was included in our analysis i.e. either the protein overlapping between studies or in case of no overlap, the protein that had the highest identified peptide count. This yielded 639 unique proteins detected in aqueous humor of either glaucoma patients or non-glaucomatous controls. In our manuscript entitled "The aqueous humor proteome of primary open angle glaucoma: An extensive review" [1], we further analysed this dataset. The dataset was exported to Perseus (version 1.6.5.0). We removed contaminants and filtered for proteins detected with high confidence, i.e. in more than 70% of the samples of at least one study. This yielded 248 proteins of which we compared the expression in glaucoma patients against control patients. Gene ontology enrichment analysis and pathway analysis was used to interpret the results. The unfiltered dataset reported in this data article and the approach reported here to reanalyse and combine raw data of different studies can be applied by other glaucoma researchers to gain more insight in the pathogenesis of glaucoma.

The aqueous humor proteome of primary open angle glaucoma: An extensive review.

BACKGROUND: We reviewed the literature on the aqueous humor (AH) proteome of primary open angle glaucoma (POAG) patients in order to obtain deeper insight into the pathophysiology of POAG. METHODS: We searched Pubmed and Embase up to May 2019 for studies that compared AH protein composition between POAG (cases) and cataract (controls). Untargeted studies (measuring the whole proteome, by LC-MS/MS) were divided into two subgroups depending on the type of surgery during which POAG AH was collected: glaucoma filtration surgery (subgroup 1) or cataract surgery (subgroup 2). We reanalyzed the raw data (subgroup 1) or combined the reported data (subgroup 2) to perform GO enrichment (GOrilla) and pathway analysis (Pathvisio). RESULTS: Out of 93 eligible proteomic studies, seven were untargeted studies that identified 863 AH proteins. We observed 73 differentially expressed proteins in subgroup 1 and 87 differentially expressed proteins in subgroup 2. Both subgroups were characterized by activation of the acute immune response, dysregulation of folate metabolism and dysregulation of the selenium micronutrient network. For subgroup 1 but not for subgroup 2, proteins of the complement system were significantly enriched. CONCLUSION: AH proteome of POAG patients shows strong activation of the immune system. In addition, analysis suggests dysregulation of folate metabolism and dysregulation of selenium as underlying contributors. In view of their glaucoma surgery, POAG patients of subgroup 1 most likely are progressive whereas POAG patients in subgroup 2 most likely have stable POAG. The proteome difference between these subgroups suggests that the complement system plays a role in POAG progression.

Stem Cell Derived Retinal Pigment Epithelium: The Role of Pigmentation as Maturation Marker and Gene Expression Profile Comparison with Human Endogenous Retinal Pigment Epithelium.

In age-related macular degeneration (AMD) the retinal pigment epithelium (RPE) deteriorates, leading to photoreceptor decay and severe vision loss. New therapeutic strategies aim at RPE replacement by transplantation of pluripotent stem cell (PSC)-derived RPE. Several protocols to generate RPE have been developed where appearance of pigmentation is commonly used as indicator of RPE differentiation and maturation. It is, however, unclear how different pigmentation stages reflect developmental stages and functionality of PSC-derived RPE cells. We generated human embryonic stem cell-derived RPE (hESC-RPE) cells and investigated their gene expression profiles at early pigmentation (EP) and late pigmentation (LP) stages. In addition, we compared the hESC-RPE samples with human endogenous RPE. We used a common reference design microarray (44 K). Our analysis showed that maturing hESC-RPE, upon acquiring pigmentation, expresses markers specific for human RPE. Interestingly, our analysis revealed that EP and LP hESC-RPE do not differ much in gene expression. Our data further showed that pigmented hESC-RPE has a significant lower expression than human endogenous RPE in the visual cycle and oxidative stress pathways. In contrast, we observed a significantly higher expression of pathways related to the process adhesion-to-polarity model that is typical of developing epithelial cells. We conclude that, in vitro, the first appearance of pigmentation hallmarks differentiated RPE. However, further increase in pigmentation does not result in much significant gene expression changes and does not add important RPE functionalities. Consequently, our results suggest that the time span for obtaining differentiated hESC-RPE cells, that are suitable for transplantation, may be greatly reduced.

The dynamic nature of Bruch's membrane.

Bruch's membrane (BM) is a unique pentalaminar structure, which is strategically located between the retinal pigment epithelium (RPE) and the fenestrated choroidal capillaries of the eye. BM is an elastin- and collagen-rich extracellular matrix that acts as a molecular sieve. BM partly regulates the reciprocal exchange of biomolecules, nutrients, oxygen, fluids and metabolic waste products between the retina and the general circulation. Accumulating evidence suggests that the molecular, structural and functional properties of BM are dependent on age, genetic constitution, environmental factors, retinal location and disease state. As a result, part of the properties of BM are unique to each human individual at a given age, and therefore uniquely affect the development of normal vision and ocular disease. The changes occurring in BM with age include increased calcification of elastic fibres, increased cross-linkage of collagen fibres and increased turnover of glycosaminoglycans. In addition, advanced glycation end products (AGEs) and fat accumulate in BM. These age-related changes may not only influence the normal age-related health of photoreceptor cells, but also the onset and progression of diseases like retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Undoubtedly, BM is the site of drusen development. Confluent drusen and uncontrolled activation of the complement cascade are most likely the first signs of AMD. Furthermore, the nature of adhesive interactions between the RPE and BM are instrumental in the development of retinal detachments and proliferative retinal disease. Finally, BM is passively or actively involved in a range of other retinal disorders such as Pseudoxanthoma elasticum (PXE), Sorsby's Fundus Dystrophy and Malattia Leventinese. Here, we review the dynamic nature of Bruch's membrane, from molecule to man, during development, aging and disease. We propose a simple and straightforward nomenclature for BM deposits. Finally, we attempt to correlate recently published mRNA expression profiles of the RPE and choroid with molecular, structural and functional properties of BM. Our review may shed light on the complex involvement of BM in retinal pathology, notably age-related macular degeneration.

[From gene to disease; pseudoxanthoma elasticum and the ABCC6 gene]. / Van gen naar ziekte; pseudoxanthoma elasticum en het ABCC6-gen.

Pseudoxanthoma elasticum (PXE) is a hereditary disease of the connective tissue characterized by progressive dystrophic mineralization of elastic fibres. PXE patients have skin lesions, may experience loss of visual acuity and cardiovascular complications. The inheritance pattern of PXE is almost always autosomal recessive. In less than 2% of the families, PXE may be inherited in an autosomal dominant fashion. PXE is caused by mutations in the ABCC6 (MRP6) gene. The R1141X mutation is by far the most common mutation; it has been identified in 19 patients, or 30% of all PXE-patients in the Netherlands. The molecular pathology of PXE is complicated by yet unknown factors causing a variable clinical expression of the disease. In 80% of the 110 PXE patients the authors studied, at least one ABCC6 mutation was found. Molecular diagnostics of PXE is especially useful to confirm the clinical diagnosis.