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Low-density lipoprotein (LDL) apheresis is effective and safe for patients with diabetes, proteinuria, and dyslipidemia. Diabetes mellitus is accompanied by ocular microvascular complications like retinal neovascularization or diabetic macular edema. These are leading causes of blindness and can be mediated by abnormal vessel growth and increased vascular permeability due to elevated levels of vascular endothelial growth factor (VEGF) in diabetic patients. In this study, we established methods to study the expression of different VEGF isoforms in human retinal and endothelial cells. The VEGF-A

Human platelet lysate (hPL) as a replacement for foetal bovine serum (FBS) in culturing human corneal endothelium is an emerging area of interest, although there are limited studies evaluating the quality of the hPL being used. Our study aimed to evaluate variations between sources of hPL and to explore the efficacy of hPL (with and without heparin) as a replacement for FBS in culturing human corneal endothelial cells in vitro. Immortalized human corneal endothelial cells (B4G12) and primary human corneal endothelial cells (PHCEnCs, n = 11 donors, age from 36 to 85 years old) were cultured with 5% hPL or FBS. A full characterisation of the effects of hPL and FBS on cell growth was conducted using IncuCyte Zoom (percentage cell confluence and population doubling time, PDT) to analyse cell proliferation. AlamarBlue assays were used to measure cell viability. The concentration of fibrinogen, PDGF, hEGF, VEGF and bFGF in two sources of hPL were analyzed by Enzyme-linked immunosorbent assay. Expression and localization of Na+/K+-ATPase, ZO-1 and CD166 on PHCEnCs and B4G12 cells were assessed with immunofluorescence and immunoblotting. Our results showed that a significant difference in fibrinogen, hEGF and VEGF concentrations was found between two sources of hPL. Heparin impaired the positive effect of hPL on cell growth. PDT and alamarBlue showed that hPL significantly increased proliferation and viability of PHCEnCs in two of three donors, and immunostaining indicated that hPL increased ZO-1 and CD166 expression but not Na+/K+-ATPase on PHCEnCs. In addition, heterogeneities on immunopositivity of Na+/K+-ATPase and ZO-1 and morphology were found on PHCEnCs derived from an individual donor cultured with hPL medium. In conclusion, hPL showed positive effect on primary corneal endothelial cell growth, and maintenance of their cellular characteristics compared to FBS. hPL can be considered as a supplement to replace FBS in PHCEnC culture. However, the variation observed between different hPL sources suggests that a standard quality control monitoring system such as storage time and a minimal concentration of growth factors may need to be established.

Corneal stromal wound healing is a well-balanced process promoted by overlapping phases including keratocyte proliferation, inflammatory-related events, and tissue remodeling. L-carnitine as a natural antioxidant has shown potential to reduce stromal fibrosis, yet the underlying pathway is still unknown. Since transient receptor potential vanilloid 1 (TRPV1) is a potential drug target for improving the outcome of inflammatory/fibrogenic wound healing, we investigated if L-carnitine can mediate inhibition of the fibrotic response through suppression of TRPV1 activation in human corneal keratocytes (HCK). We determined TRPV1-induced intracellular calcium transients using fluorescence calcium imaging, channel currents by planar patch-clamping, and cell migration by scratch assay for wound healing. The potential L-carnitine effect on TRPV1-induced myofibroblast transdifferentiation was evaluated by immunocytochemical detection of alpha smooth muscle actin. RT-PCR analysis confirmed TRPV1 mRNA expression in HCK. L-carnitine (1 mmol/l) inhibited either capsaicin (CAP) (10 µmol/l), hypertonic stress (450 mOsmol/l), or thermal increase (>43 °C) induced Ca2+ transients and corresponding increases in TRPV1-induced inward and outward whole-cell currents. This was accompanied by suppression of injury-induced increases in myofibroblast transdifferentiation and cell migration. In conclusion, L-carnitine contributes to inhibit stromal scarring through suppressing an injury-induced intrinsic TRPV1 activity that is linked with induction of myofibroblast transdifferentiation in HCK cells., Human corneal keratocytes (HCK) are essential for maintaining corneal structure and transparency. This study shows that the natural antioxidant L-carnitine inhibits stromal scarring by suppressing injury-induced intrinsic transient receptor potential vanilloid type 1 (TRPV1) activity that is linked to induction of myofibroblast transdifferentiation in HCK cells. Blocking TRPV1 activation on keratocytes may therefore be a viable approach to suppress corneal opacification in a clinical setting.

Intracellular formation of advanced glycation end products (AGEs) is a crucial pathological process in retinal diseases such as age-related macular degeneration (AMD) or diabetic retinopathy (DR). Glyoxal is a physiological metabolite produced during formation of AGEs and has also been shown to derive from photodegraded bisretinoid fluorophores in aging retinal pigment epithelial (RPE) cells.Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of NIncreasing glyoxal concentrations resulted in enhanced formation of AGEs, such as CML modifications of proteins. This was associated with elevated levels of intracellular reactive oxygen species, a depolarized MMP, and a decreased intracellular pH, resulting in an increased number of apoptotic cells. Apoptosis related caspase 3 activation increased in a dose dependent manner after glyoxal incubation. In consequence, the cells activated compensatory mechanisms and increased the levels of the anti-oxidative and stress-related proteins heme oxygenase-1, osteopontin, heat shock protein 27, copper/zinc superoxide dismutase, manganese superoxide dismutase, and cathepsin D. Furthermore, VEGF-AThe glyoxal-induced oxidative stress and apoptosis in ARPE-19 cells may provide a suitable in vitro model for studying RPE cellular reactions to AGEs that occur in AMD or in DR.

Das Makulaforamen (MF) ist eine Erkrankung des Sehzentrums der Netzhaut des menschlichen Auges und fuhrt unbehandelt zu starker Sehbeeintrachtigung. Die Makulaforamenchirurgie ist seit Jahren sehr erfolgreich und zeigt in fruhen Stadien einen sehr guten anatomischen und funktionellen Erfolg, weniger jedoch – trotz standiger Verbesserung der Operationsprozeduren – in hoheren Erkrankungsstadien. In einer retrospektiven Analyse untersuchten wir die Wirkung von autologem Thrombozytenkonzentrat bei Patienten mit einem hohen MF-Stadium (Stadium III–IV) hinsichtlich des Lochverschlusses und der Erholung der Sehkraft (Visus). Die Applikation der Thrombozyten erfolgte intraoperativ bei einer pars plana Vitrektomie (ppV). Zusatzlich wurden die Plattchenkonzentrate bei einigen Patienten qualitativ auf den Gehalt an Wachstumsfaktoren und mit Blick auf ihre Adhasionsfahigkeit untersucht. In der ersten Patientengruppe konnte ein guter anatomischer Lochverschluss bei 74 % der Patienten erzielt werden. Die Analysen der Plattchenkonzentrate in dieser Gruppe zeigten, dass ein moglicher Wundheilungseffekt am ehesten vom Gehalt an Wachstumsfaktoren (am Beispiel des Plattchenwachstumsfaktors, PDGF) beeinflusst wird und weniger von der Adharenzfahigkeit der Thrombozyten, gemessen an der Aggregationsfahigkeit nach Ristocetingabe. In der zweiten Patientengruppe zeigten sich nach Optimierung des Verfahrens fur die Thrombozytenkonzentratherstellung und des chirurgischen Verfahrens eine Steigerung des anatomischen Erfolgs (92 %) sowie eine sehr rasche Entwicklung der Visusverbesserung innerhalb der ersten sechs Wochen. Die Optimierung der chirurgischen Verfahren zur MF-Chirurgie stand in der Vergangenheit an erster Stelle. Konzepte zur Wundheilung sind eher rar. Wir postulieren aufgrund unserer Daten, dass der Einsatz von autologen Thrombozytenkonzentraten als Wundheilungskonzept gerade den funktionellen Erfolg in spaten Stadien der Erkrankung steigern hilft.

The macular hole (MH) is a disorder of the visual center of the retina in humans. An untreated MH leads to loss of central visual acuity and reading ability. Surgery for early-stage macular holes has been very successful for many years and leads to very good anatomical and functional results. Despite continuous improvement of surgical procedures, the outcome for the later stages of MH is still unsatisfactory.In a retrospective analysis, we investigated the effect of autologous platelet concentrates in patients presenting later stages of MHs (stage III-IV) with respect to anatomic success (hole closure) and recovery of vision. The application of platelets was performed during retinal surgery (pars plana vitrectomy, ppV). In addition, selected platelet concentrates were qualitatively analysed for growth factors and platelet adhesion.In the first group, 74% of the patients showed a good anatomical macular hole closure. The analyses of the platelet concentrates indicated a possible wound-healing effect due to growth factors (e.g. the platelet-derived growth factor, PDGF) and lesser to the ability of the platelets to adhere after ristocetin administration. Further optimization of the production process of platelet concentrates and of the surgical procedure in the second group of patients showed an increase of the anatomical success (92%) and a very rapid increase of visual acuity within six weeks.In the past, the primary goal of MH surgery was to optimize the surgical procedures. Only few concepts focused on wound healing. Based on our data, we postulate the use of autologous platelet concentrates in MH surgery as a healing concept, which helps to increase the functional success of late-stage macular hole surgery.

Two established material systems for thermally stimulated detachment of adherent cells were combined in a cross-linked polymer blend to merge favorable properties. Through this approach poly(N-isopropylacrylamide) (PNiPAAm) with its superior switching characteristic was paired with a poly(vinyl methyl ether)-based composition that allows adjusting physico-chemical and biomolecular properties in a wide range. Beyond pure PNiPAAm, the proposed thermo-responsive coating provides thickness, stiffness and swelling behavior, as well as an apposite density of reactive sites for biomolecular functionalization, as effective tuning parameters to meet specific requirements of a particular cell type regarding initial adhesion and ease of detachment. To illustrate the strength of this approach, the novel cell culture carrier was applied to generate transplantable sheets of human corneal endothelial cells (HCEC). Sheets were grown, detached, and transferred onto planar targets. Cell morphology, viability and functionality were analyzed by immunocytochemistry and determination of transepithelial electrical resistance (TEER) before and after sheet detachment and transfer. HCEC layers showed regular morphology with appropriate TEER. Cells were positive for function-associated marker proteins ZO-1, Na+/K+-ATPase, and paxillin, and extracellular matrix proteins fibronectin, laminin and collagen type IV before and after transfer. Sheet detachment and transfer did not impair cell viability. Subsequently, a potential application in ophthalmology was demonstrated by transplantation onto de-endothelialized porcine corneas in vitro. The novel thermo-responsive cell culture carrier facilitates the generation and transfer of functional HCEC sheets. This paves the way to generate tissue engineered human corneal endothelium as an alternative transplant source for endothelial keratoplasty.