Pathogenese des Keratokonus – inflammatorische Komponente durch metabolische Veränderungen?

Der Keratokonus (KC) ist eine progressive Erkrankung der Hornhaut. Beim Keratokonus wird die Hornhaut dünner und wölbt sich konisch nach vorne. Die Erkrankung tritt in der Regel im Teenageralter auf. Etwa 1 von 2000 aus der Bevölkerung sind in Europa von der Erkrankung betroffen. Bei einem Teil der Patienten verlangsamt sich die Erkrankung oder die Progression stoppt, bei einem Teil schreitet die Progression immer weiter. Die Behandlung erfolgt zunächst mit harten Kontaktlinsen, um die nachlassende Sehschärfe auszugleichen. Für einige Patienten kommen das Riboflavin-Crosslinking oder die Implantation intrakornealer Ringsegmente als Behandlungsmethode in Frage, um die Progression der Erkrankung zu stoppen. Für einen Teil der Patienten bleibt nur noch die Hornhauttransplantation, um die Sehschärfe zu verbessern. In der ersten Arbeit wurden Harnstoff, Harnsäure, Prolaktin und fT4 im Kammerwasser von Keratokonuspatienten untersucht. Es konnte gezeigt werden, dass die Harnstoff und Prolaktin Konzentrationen im Kammerwasser von KC-Patienten erniedrigt sind, während fT4 erhöht ist. Da der Harnstoffzyklus bei der Biosynthese von Aminosäuren im Sinne der Kollagensynthese eine entscheidende Rolle spielt, wurde dieser Parameter als Ausgangspunkt für weitere Studien angesehen. In der zweiten Studie wurde in Zellkulturversuchen gezeigt, dass die Harnstoff Konzentration auch in den Keratozyten von KC-Patienten reduziert ist. Da Harnstoff ein Nebenprodukt bei der Synthese von Prolin und Hydroxyprolin ist, haben wir den Harnstoffzyklus in den Keratozyten genauer untersucht. Die Ergebnisse dieser Arbeit lieferten weitere Hinweise auf eine veränderte Regulation der Kollagensynthese in KC-Keratozyten. Die Studie ergab, dass die Keratokonus Keratozyten eine erniedrigte Arginase Aktivität und eine erniedrigte Hydroxyprolin Konzentration im Vergleich zu den gesunden Kontrollzellen aufweisen. Die Ergebnisse der dritten Arbeit unterstützen die These einiger Forschungsgruppen, dass die Krankheit mit entzündlichen Prozessen einhergeht. Ein Schlüssel-Gen bei entzündlichen Prozessen ist der Nuklear Faktor kappa B (NF-κB). Als Transkriptionsfaktor aktiviert NF-κB unter anderem das bei entzündlichen Prozessen mitwirkende Enzym Nitric Oxide Synthase (NOS). Die Ergebnisse dieser Arbeit zeigten eine erhöhte NF-κB mRNA und Protein Expression, sowie auch eine erhöhte Stickoxidproduktion und induzierte NOS mRNA Expression in Keratokonus Keratozyten. Weitere darauf aufbauende Untersuchungen sind notwendig, um mehr Einblick in die Erkrankung und damit auch die Möglichkeit neuer Behandlungsmethoden für die Patienten durch weitere Erkenntnisse hinsichtlich der Pathogenese zu gewinnen. Pathogenesis of Keratoconus – Inflammatory components through metabolic changes? Keratoconus (KC) is a progressive corneal disease, which is characterized by corneal thinning and conical shape. It usually appears in teenagers and about 1 of 2000 persons is affected in Europe. In some of the patients progression is getting slower with age and then it stops, in some others it accelerates. The stage-related treatment starts with rigid gas permeable contact lenses to compensate corneal irregularities and to improve vision. In case of progression, riboflavin crosslinking or implantation of intracorneal ring segments may be used to stabilize the cornea. In some of the patients, corneal transplantation is performed in order to improve visual acuity. In the first paper urea, uric acid, prolactin and fT4 concentration were examined in aqueous humor of keratoconus patients. It could be shown that urea and prolactin concentrations are decreased and fT4 concentration is increased in aqueous humor of keratoconus patients. Since the urea cycle plays a decisive role in the biosynthesis of non-essential proteinogenic amino acids in terms of collagen synthesis, this parameter was considered as a crucial starting point for further studies. In the second study, a reduced urea concentration could be verified in keratoconus keratocyte cultures. Since urea is a by-product in the synthesis of proline and hydroxyproline, we examined the urea cycle in keratocytes more closely. The results of this work provided further evidence for altered regulation of collagen synthesis in KC-keratocytes. Here, a decreased arginase activity and hydroxyproline concentration could be shown in keratoconus keratocytes, compared to normal controls. The results of the third study supported the hypothesis of some research groups, that keratoconus is associated with inflammatory processes. A key gene in inflammatory processes is the nuclear factor kappa B (NF-κB). As a transcription factor, NF-κB activates the enzyme Nitric Oxide Synthase (NOS), which is involved in inflammatory processes. The results of this work showed an increased NF-κB mRNA and protein expression, as well as increased nitric oxide production and induced NOS mRNA expression in keratoconus keratocytes. Further investigations based on these results are necessary in order to gain more insight into the pathogenesis of keratoconus and thus, to new treatment modalities for the patients.

Hypoxic stress increases NF-κB and iNOS mRNA expression in normal, but not in keratoconus corneal fibroblasts

Background Keratoconus (KC) is associated with oxidative stress and hypoxia and as several times discussed, potentially with inflammatory components. Inflammation, hypoxia, and oxidative stress may result in metabolic dysfunction and are directly linked to each other. In the current study, we investigate the effect of hypoxia through NF-κB signaling pathways on iNOS, hypoxia-induced factors (HIF), ROS, and proliferation of normal and KC human corneal fibroblasts (HCFs), in vitro. Methods Primary human KC-HCFs and normal HCFs were isolated and cultured in DMEM/Ham’s F12 medium supplemented with 5% fetal calf serum. Hypoxic conditions were generated and quantitative PCR and Western blot analysis were performed to examine NF-κB, iNOS, HIF, and PHD2 expression in KC and normal HCFs. ROS level was analyzed using flow cytometry and proliferation by BrdU-ELISA. Results Hypoxia increased NF-κB mRNA and protein expression in normal HCFs, but in KC-HCFs NF-κB mRNA and protein expression remained unchanged. Hypoxic conditions upregulated iNOS mRNA expression of normal HCFs, but iNOS mRNA expression of KC-HCFs and iNOS protein expression of both HCF types remained unchanged. Hypoxia downregulated HIF-1α and HIF-2α mRNA expression in normal and KC-HCFs. PHD2 mRNA expression is upregulated under hypoxia in KC-HCFs, but not in normal HCFs. PHD2 protein expression was upregulated by hypoxia in both HCF types. Total ROS concentration is downregulated in normal and KC-HCFs under hypoxic conditions. Proliferation rate of KC-HCFs was upregulated through hypoxia, but did not change in normal HCFs. Conclusions Hypoxia increases NF-κB and iNOS mRNA expression in normal HCFs, but there does not seem to be enough capacity in KC-HCFs to increase NF-κB and iNOS mRNA expression under hypoxia, maybe due to the preexisting oxidative stress. HIF and PHD2 do not show altered iNOS regulation under hypoxic conditions in KC-HCFs, and therefore do not seem to play a role in keratoconus pathogenesis. An increased proliferation of cells may refer to compensatory mechanisms under hypoxia in KC. Understanding the mechanism of the altered regulation of NF-κB and iNOS in KC-HCFs will provide better insight into the potential inflammatory component of the KC pathogenesis

NF-κB, iNOS, IL-6, and collagen 1 and 5 expression in healthy and keratoconus corneal fibroblasts after 0.1% riboflavin UV-A illumination

Purpose To analyze the effect of riboflavin UV-A illumination on mRNA and protein expression of healthy (HCFs) and keratoconus human corneal fibroblasts (KC-HCFs), concerning the inflammatory markers NF-κB, iNOS, IL-6, and collagen 1 and 5 (Col 1/Col 5). Methods Keratocytes were isolated from healthy (n = 3) and keratoconus (KC) corneas (n = 3) and were cultivated in basal medium with 5% fetal calf serum, which resulted in their transformation into human corneal fibroblasts (HCFs/KC-HCFs). Cells underwent 0.1% riboflavin UV-A illumination for 250 s (CXL). NF-κB, iNOS, IL-6, Col 1, and Col 5 expression was investigated by qPCR and Western blot analysis. IL-6 concentration of the cell culture supernatant and cell lysate was determined by ELISA. Results In untreated KC-HCFs, NF-κB (p = 0.0002), iNOS (p = 0.0019), Col 1 (p = 0.0286), and Col 5 (p = 0.0054) mRNA expression was higher and IL-6 expression was lower (p = 0.0057), than in healthy controls. In HCFs, CXL led to an increased NF-κB (p = 0.0286) and IL-6 (p = 0.0057) mRNA expression. The IL-6 concentration in the cell culture supernatant was increased in HCFs (p = 0.0485) and KC-HCFs (p = 0.0485) after CXL. CXL increased intracellular IL-6 concentration only in KC-HCFs (p = 0.0357). In the HCF group (p = 0.0286), an increased Col 1 mRNA expression after CXL could be observed. Conclusion Our study confirmed altered gene expression in untreated KC-HCFs compared to untreated HCFs. Riboflavin UV-A illumination affected gene expression only in HCFs. Increased IL-6 concentration in the cell culture supernatant and cell lysate indicate a secondary inflammatory response of HCFs and KC-HCFs to riboflavin UV-A illumination

Keratin 12 mRNA expression could serve as an early corneal marker for limbal explant cultures

This investigation aimed to identify early corneal marker and conjunctival epithelial differentiation through transcriptional analysis of limbal explant cultures and study early differentiation patterns of known corneal and conjunctival differentiation markers. 2 mm punch biopsies of limbal region were obtained from 6 donors of the Lions Cornea Bank Saar-Lorloux/Trier-Westpfalz. Limbal explants were dissected into corneal and conjunctival biopsy sections. Biopsies were placed with epithelial side down into 12 Wells. As soon as the outgrowing cells had reached confluence, they were harvested. mRNA expression of corneal differentiation markers KRT12, KRT3, DSG1, PAX6, ADH7 and ALDH1A1, conjunctival markers KRT19, KRT13 and stem cell marker ABCG2 were measured via qPCR. KRT12 and PAX6 protein expressions were evaluated using Western Blot. Results suggested that KRT12 mRNA expression was significantly higher in outgrowing cells from the corneal side of the biopsies as in those from the conjunctival side (p = 0.0043). There was no significant difference in mRNA expression of other analyzed markers comparing with marker expression of outgrown cells from both limbal biopsies (p > 0.13). KRT12 and PAX6 Western Blot analysis showed no difference in cells harvested from both sides. In conclusion, KRT12 mRNA might be a marker to measure corneal origin of cells from limbal biopsies with unknown composition of corneal and conjunctival progenitor cells. KRT3, DSG1, PAX6, ADH7, ALDH1A1, KRT19, KRT13 and ABCG2 mRNA as well as KRT12 and PAX6 protein expression could not contribute to differentiate corneal from conjunctival cell identity from limbal biopsies

Altered Regulation of mRNA and miRNA Expression in Epithelial and Stromal Tissue of Keratoconus Corneas

Purpose: Evaluation of mRNA and microRNA (miRNA) expression in epithelium and stroma of patients with keratoconus. Methods: The epithelium and stroma of eight corneas of eight patients with keratoconus and eight corneas of eight non-keratoconus healthy controls were studied separately. RNA was extracted, and mRNA and miRNA analyses were performed using microarrays. Differentially expressed mRNAs and miRNAs in epithelial and stromal keratoconus samples compared to healthy controls were identified. Selected genes and miRNAs were further validated using RT-qPCR. Results: We discovered 170 epithelial and 1498 stromal deregulated protein-coding mRNAs in KC samples. In addition, in epithelial samples 180 miRNAs and in stromal samples 379 miRNAs were significantly deregulated more than twofold compared to controls. Pathway analysis revealed enrichment of metabolic and axon guidance pathways for epithelial cells and enrichment of metabolic, mitogen-activated protein kinase (MAPK), and focal adhesion pathways for stromal cells. Conclusions: This study demonstrates significant differences in the expression and regulation of mRNAs and miRNAs in the epithelium and stroma of Patients with KC. Also, in addition to the well-known target candidates, we were able to identify further genes and miRNAs that may be associated with keratoconus. Signaling pathways influencing metabolic changes and cell contacts are affected in epithelial and stromal cells of patients with keratoconus

Herpes simplex virus PCR in 2230 explanted corneal buttons

Purpose: To determine herpes simplex virus (HSV) DNA prevalence and mean cycle threshold of polymerase chain reaction (PCR) in corneal tissue of patients with penetrating keratoplasty (PKP), with (HSK+) and without (HSK−) previous clinical herpetic keratitis history. Methods: Retrospective review of recipient corneal buttons which were explanted through PKP between March 2010 and September 2018 at the Department of Ophthalmology, Saarland University Medical Center in Homburg/Saar, Germany. Corneal tissue samples were analysed by real-time PCR for the presence of HSV DNA. For each subject, clinical data, including patients’ demographics and clinical diagnoses, were collected. Results: In total, 2230 corneal samples (age at the time of the surgery 57.3 19.2 years) of 1860 patients were analysed. HSV PCR was positive in 137 (6.1%) corneal samples, with a 30.57 6.01 (range 14–39) mean cycle threshold (Ct) value. Two hundred ninety-eight (13.4%) corneas of 266 patients were clinically HSK+, and 1932 (86.6%) corneas of 1600 patients were clinically HSK−. HSV DNA was detected significantly more frequently (p < 0.0001) in HSK+ corneal samples (108 corneal samples; 36.2%), than in HSK− corneal samples (29 corneal samples; 1.5%). Ct value was significantly lower in HSK+ than in HSK- corneal samples (29.8 5.8 versus 32.6 5.9; p = 0.008). Conclusion: Our data demonstrate that a positive clinical history of HSK is related to HSV PCR positivity in about every 2.8th patient. In addition, about every 66th explanted corneal tissue is HSV PCR-positive despite the lack of clinical suspicion. These patients may need additional local/systemic antiviral treatment to avoid newly acquired HSK following penetrating keratoplasty

Similarities in DSG1 and KRT3 Downregulation through Retinoic Acid Treatment and PAX6 Knockdown Related Expression Profiles: Does PAX6 Affect RA Signaling in Limbal Epithelial Cells?

Congenital PAX6-aniridia is a rare panocular disease resulting from limbal stem cell defi ciency. In PAX6-aniridia, the downregulation of the retinol-metabolizing enzymes ADH7 (All-trans retinol dehydrogenase 7) and ALDH1A1/A3 (Retinal dehydrogenase 1, Aldehyde dehydrogenase family 1 member A3) have been described in limbal epithelial cells (LECs) and conjunctival epithelial cells. The aim of this study was to identify the role of retinol derivates in the differentiation of human LEC and its potential impact on aniridia-associated keratopathy development. Human LEC were isolated from healthy donor corneas and were cultured with retinol, retinoic acid, or pan-retinoic acid receptor antagonist (AGN 193109) acting on RARα, β, γ (NR1B1, NR1B2 NR1B3) or were cultured with pan-retinoid X receptor antagonist (UVI 3003) acting on RXR α, β, γ (retinoid X receptor, NR2B1, NR2B2, BR2B3). Using qPCR, differentiation marker and retinoid-/fatty acid metabolism-related mRNA expression was analysed. DSG1 (Desmoglein 1), KRT3 (Keratin 3), and SPINK7 (Serine Pepti dase Inhibitor Kazal Type 7) mRNA expression was downregulated when retinoid derivates were used. AGN 193109 treatment led to the upregulation of ADH7, KRT3, and DSG1 mRNA expression and to the downregulation of KRT12 (Keratin 12) and KRT19 (Keratin 19) mRNA expression. Retinol and all-trans retinoic acid affect some transcripts of corneal LEC in a similar way to what has been observed in the LEC of PAX6-aniridia patients with the altered expression of differentiation markers. An elevated concentration of retinol derivatives in LEC or an altered response to retinoids may contribute to this pattern. These initial findings help to explain ocular surface epithelia differentiation disorders in PAX6-aniridia and should be investigated in patient cells or in cell models in the future in more detail

Efficacy of Off-Label Anti-Amoebic Agents to Suppress Trophozoite Formation of Acanthamoeba spp. on Non-Nutrient Agar Escherichia Coli Plates

Acanthamoeba keratitis (AK) is a dangerous infectious disease, which is associated with a high risk of blindness for the infected patient, and for which no standard therapy exists thus far. Patients suffering from AK are thus treated, out of necessity, with an off-label therapy, using drugs designed and indicated for other diseases/purposes. Here, we tested the capability of the off-label anti-amoebic drugs chlorhexidine (CH; 0.1%), dibromopropamidine diisethionate (DD; 0.1%), hexamidine diisethionate (HD; 0.1%), miltefosine (MF; 0.0065%), natamycin (NM; 5%), polyhexamethylene biguanide (PHMB; 0.02%), povidone iodine (PVPI; 1%), and propamidine isethionate (PD; 0.1%) to suppress trophozoite formation of Acantamoeba castellanii and Acanthamoeba hatchetti cysts on non-nutrient agar Escherichia coli plates. Of the eight off-label anti-amoebic drugs tested, only PVPI allowed for a complete suppression of trophozoite formation by drug-challenged cysts for all four Acanthamoeba isolates in all five biological replicates. Drugs such as NM, PD, and PHMB repeatedly suppressed trophozoite formation with some, but not all, tested Acanthamoeba isolates, while other drugs such as CH, DD, and MF failed to exert a relevant effect on the excystation capacities of the tested Acanthamoeba isolates in most, if not all, of our repetitions. Our findings suggest that pre-testing of the AK isolate with the non-nutrient agar E. coli plate assay against the anti-amoebic drug intended for treatment should be performed to confirm that the selected drug is cysticidal for the Acanthamoeba isolate

Identification of the regulatory circuit governing corneal epithelial fate determination and disease.

The transparent corneal epithelium in the eye is maintained through the homeostasis regulated by limbal stem cells (LSCs), while the nontransparent epidermis relies on epidermal keratinocytes for renewal. Despite their cellular similarities, the precise cell fates of these two types of epithelial stem cells, which give rise to functionally distinct epithelia, remain unknown. We performed a multi-omics analysis of human LSCs from the cornea and keratinocytes from the epidermis and characterized their molecular signatures, highlighting their similarities and differences. Through gene regulatory network analyses, we identified shared and cell type-specific transcription factors (TFs) that define specific cell fates and established their regulatory hierarchy. Single-cell RNA-seq (scRNA-seq) analyses of the cornea and the epidermis confirmed these shared and cell type-specific TFs. Notably, the shared and LSC-specific TFs can cooperatively target genes associated with corneal opacity. Importantly, we discovered that FOSL2, a direct PAX6 target gene, is a novel candidate associated with corneal opacity, and it regulates genes implicated in corneal diseases. By characterizing molecular signatures, our study unveils the regulatory circuitry governing the LSC fate and its association with corneal opacity