Increased Angiopoietin-1 and -2 levels in human vitreous are associated with proliferative diabetic retinopathy

$\bf Background$ Diabetic retinopathy is a frequent complication of diabetes mellitus and a leading cause of blindness in adults. The objective of this study was to elucidate the diabetic retinopathy pathophysiology in more detail by comparing protein alterations in human vitreous of different diabetic retinopathy stages. $\bf Methods$ Vitreous samples were obtained from 116 patients undergoing pars plana vitrectomy. Quantitative immunoassays were performed of angiogenic factors (VEGF-A, PIGF, Angiopoietin-1, Angiopoietin-2, Galectin-1) as well as cytokines (IL-1$\beta$, IL-8, IFN-$\gamma$, TNF-$\alpha$, CCL3) in samples from control patients (patients who don’t suffer from diabetes; n = 58) as well as diabetes mellitus patients without retinopathy (n = 25), non-proliferative diabetic retinopathy (n = 12), and proliferative diabetic retinopathy patients (n = 21). In addition, correlation analysis of protein levels in vitreous samples and fasting glucose values of these patients as well as correlation analyses of protein levels and VEGF-A were performed. $\bf Results$ We detected up-regulated levels of VEGF-A (p = 0.001), PIGF (p<0.001), Angiopoietin-1 (p = 0.005), Angiopoietin-2 (p<0.001), IL-1$\beta$ (p = 0.012), and IL-8 (p = 0.018) in proliferative diabetic retinopathy samples. Interestingly, we found a strong positive correlation between Angiopoietin-2 and VEGF-A levels as well as a positive correlation between Angiopoietin-1 and VEGF-A. $\bf Conclusion$ This indicated that further angiogenic factors, besides VEGF, but also pro-inflammatory cytokines are involved in disease progression and development of proliferative diabetic retinopathy. In contrast, factors other than angiogenic factors seem to play a crucial role in non-proliferative diabetic retinopathy development. A detailed breakdown of the pathophysiology contributes to future detection and treatment of the disease

Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients

Evaluation of cytokines in patients with diabetic retinopathy (DR) is important for the identification of future additive or alternative treatment options. Therefore, vitreous samples were obtained from patients with DR and patients with macular hole or macular pucker (control group) during 23-gauge-vitrectomy (n = 17/group). The levels of three pro-inflammatory (IL-1$\beta$, IL-6, IFN-$\gamma$) and pleiotropic cytokines (IL-2, IL-4, IL-13) as well as VEGF, VEGF-A, and PGF were measured using an enzyme linked immunosorbent assay (ELISA). IL-1$\beta$ (p = 0.02) and IFN-$\gamma$ (p = 0.04), two of the three tested pro-inflammatory cytokines, were elevated in the DR patients, while IL-6 (p = 0.51) level was comparable in both groups. Moreover, in DR samples, a trend towards an IL-13 down-regulation (p = 0.36) was observable. The IL-2 (p = 0.62) and IL-4 (p = 0.78) levels were comparable in both groups. All analyzed angiogenetic factors were up-regulated in DR patients (VEGF: p<0.001; VEGF-A: p = 0.002; PGF: p<0.001). The up-regulation of angiogenetic factors underline their importance in DR development. However, the interaction of the other cytokines showed an interesting pattern. Pro-inflammatory cytokines were also up-regulated, which could be evidence for inflammation processes in the diabetic retina. Furthermore, it seems that a counter response of immunomodulatory cytokines is in an initial process, but not strong enough to regulate the processes. Therefore, to support these anti-inflammatory mechanisms might be additive treatment option in the future

Protein profiling of WERI-RB1 and etoposide-resistant WERI-ETOR reveals new insights into topoisomerase inhibitor resistance in retinoblastoma

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q 2|, 22 higher in WERI-ETOR). Pathway analysis revealed the "retinoid metabolism and transport" pathway as an enriched metabolic pathway in WERI-ETOR cells, while the "sphingolipid de novo biosynthesis" pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of "sphingolipid de novo biosynthesis" in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB

Expression changes and impact of the extracellular matrix on etoposide resistant human retinoblastoma cell lines

Retinoblastoma (RB) represents the most common malignant childhood eye tumor worldwide. Several studies indicate that the extracellular matrix (ECM) plays a crucial role in tumor growth and metastasis. Moreover, recent studies indicate that the ECM composition might influence the development of resistance to chemotherapy drugs. The objective of this study was to evaluate possible expression differences in the ECM compartment of the parental human cell lines WERI-RB1 (retinoblastoma 1) and Y79 and their Etoposide resistant subclones via polymerase chain reaction (PCR). Western blot analyses were performed to analyze protein levels. To explore the influence of ECM molecules on RB cell proliferation, death, and cluster formation, WERI-RB1 and resistant WERI-ETOR cells were cultivated on Fibronectin, Laminin, Tenascin-C, and Collagen IV and analyzed via time-lapse video microscopy as well as immunocytochemistry. We revealed a significantly reduced mRNA expression of the proteoglycans $\it Brevican$, $\it Neurocan$, and $\it Versican$ in resistant WERI-ETOR compared to sensitive WERI-RB1 cells. Also, for the glycoproteins $\it α1-Laminin$, $\it Fibronectin$, $\it Tenascin-C$, and $\it Tenascin-R$ as well as $\it Collagen IV$, reduced expression levels were observed in WERI-ETOR. Furthermore, a downregulation was detected for the matrix metalloproteinases $\it MMP2$, $\it MMP7$, $\it MMP9$, the tissue-inhibitor of metalloproteinase $\it TIMP2$, the Integrin receptor subunits $\it ITGA4$, $\it ITGA5$ and $\it ITGB1$, and all $\textit {receptor protein tyrosine phosphatase}$ $\beta$/$\zeta$ isoforms. Downregulation of $\it Brevican$, $\it Collagen IV$, $\it Tenascin-R$, $\it MMP2$, $\it TIMP2$, and $\it ITGA5$ was also verified in Etoposide resistant Y79 cells compared to sensitive ones. Protein levels of Tenascin-C and MMP-2 were comparable in both WERI cell lines. Interestingly, Fibronectin displayed an apoptosis-inducing effect on WERI-RB1 cells, whereas an anti-apoptotic influence was observed for Tenascin-C. Conversely, proliferation of WERI-ETOR cells was enhanced on Tenascin-C, while an anti-proliferative effect was observed on Fibronectin. In WERI-ETOR, cluster formation was decreased on the substrates Collagen IV, Fibronectin, and Tenascin-C. Collectively, we noted a different ECM mRNA expression and behavior of Etoposide resistant compared to sensitive RB cells. These findings may indicate a key role of ECM components in chemotherapy resistance formation of RB

Zur Versorgungssituation und konservativen Therapie des okulären vernarbenden Schleimhautpemphigoids in Deutschland

$\bf Hintergrund:$ Die okuläre Beteiligung bei vernarbendem Schleimhautpemphigoid (SHP) ist mit einer Prävalenz von 25 Fällen je 1 Mio. Einwohner und damit ca. 2100 Patienten in ganz Deutschland selten. Die Diagnosestellung kann – besonders in Abwesenheit anderer Beteiligungen – schwierig und die Therapie komplex und langwierig sein. Nicht selten kommen Immunsuppressiva zum Einsatz. Aufgrund der Komplexität von Diagnose und Therapie sind SHP-Patienten meist an entsprechend spezialisierte Zentren angebunden. Ziel dieses Projektes war die Erfassung der aktuellen augenärztlichen Versorgungssituation von Patienten mit SHP in Deutschland. $\bf Methoden:$ Eine papierbasierte Umfrage wurde konzipiert und im April 2020 an alle Universitätsaugenkliniken und weitere potenzielle Zentren versandt. Gefragt wurde nach dem Bestehen einer spezialisierten Sprechstunde, der jährlichen Gesamtzahl der betreuten Patienten, der jährlichen Anzahl von neu diagnostizierten Patienten, den klinischen Kooperationspartnern in Diagnostik und Therapie sowie nach der angewendeten lokalen und systemischen Therapie. $\bf Ergebnisse:$ Von insgesamt 44 angeschrieben Kliniken erfolgten 28 (64%) vollständige Rückmeldungen. Im Mittel werden in den Kliniken 27 $\pm$ 42 (0–200) Patienten betreut und jährlich pro Zentrum 3,6 $\pm$ 2,2 (0–10) neue Fälle diagnostiziert. Dies entspricht einer Gesamtpatientenzahl von 741 Patienten. Lediglich 9 (32%) der antwortenden Kliniken bieten eine spezialisierte SHP-Sprechstunde an. 93% der Zentren kooperieren mit der lokalen Klinik für Dermatologie. 79% führen die serologische und histologische Diagnostik intern durch. Etwa die Hälfte der Zentren (n = 16) wendet ein standardisiertes Therapieschema an. Systemisch werden Glukokortikoide (66,7%) am häufigsten verwendet, gefolgt von Mycophenolatmofetil und Dapson (57,1 %), Rituximab (33,3%), Azathioprin und Cyclophosphamid (28,6%) sowie Methotrexat (19,0%). Am seltensten werden i.v. Immunoglobuline eingesetzt (14,3 %). $\bf Schlussfolgerung:$ Mit dieser Umfrage unter deutschen augenärztlichen Zentren wurden Daten von etwa einem Drittel der geschätzten Gesamtzahl aller in Deutschland an einem SHP erkrankten Menschen erhoben. Dabei handelt es sich vermutlich ausschließlich um Patienten mit mindestens einer okulären Beteiligung. Aktuell wird eine augenärztliche SHP-Registerstudie etabliert, um die Epidemiologie und Versorgungssituation besser zu erfassen und langfristig zu verbessern