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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.

Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca(2+) transients induced by either menthol (500 μmol/l), eucalyptol (3 mmol/l), or icilin (2-60 μmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La(3+), 100 μmol/l) as well as the TRPM8 blockers BCTC (10 μmol/l) and capsazepine (CPZ, 10 μmol/l) suppressed icilin-induced Ca(2+) increases. In and outward currents induced by application of menthol (500 μmol/l) or icilin (50 μmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca(2+) increases that were inhibited by a TRPM8 blocker BCTC (10 μmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca(2+) response patterns are suggestive of other Ca(2+) handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype.

Functional impairment of the human corneal endothelium can lead to corneal blindness. In order to meet the high demand for transplants with an appropriate human corneal endothelial cell density as a prerequisite for corneal function, several tissue engineering techniques have been developed to generate transplantable endothelial cell sheets. These approaches range from the use of natural membranes, biological polymers and biosynthetic material compositions, to completely synthetic materials as matrices for corneal endothelial cell sheet generation. This review gives an overview about currently used materials for the generation of transplantable corneal endothelial cell sheets with a special focus on thermo-responsive polymer coatings.

The transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+)-and Mg(2+) permeable cation channel that might be a cellular osmosensor since it is activated upon hypotonic cell swelling. TRPV4 is also thermosensitive and responds to moderate heat (from 24 to 27 °C) as well as to phorbol esters (4α-PDD) and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. The resulting Ca(2+) influx occurring in response to swelling induces regulatory volume decrease (RVD) behavior. As regulation of cell volume is essential for corneal endothelial function, we determined whether human corneal endothelial cells have functional TRPV4 channel activity. RT-PCR identified TRPV4 gene expression in the HCEC-12 cell line as well as two clonal daughter cell lines (HCEC-H9C1, HCEC-B4G12). [Ca(2+)](i) transients were monitored in fura-2 loaded cells. Nonselective cation channel currents were recorded in the whole-cell mode of the planar patch-clamp technique. TRPV4 mRNA was found in HCEC-12 and the clonal daughter cell lines. TRPV4 channel agonists (4α-PDD and GSK1016790A; both 5 μmol/l) as well as moderate heat (

The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light-emitting diode (LED) modules with peaks at either 411nm (half bandwidth 17nm) or 470nm (half bandwidth 25nm) at defined irradiances of 0.6, 1.5 and 4.5W/m(2) for 411nm and 4.5W/m(2) for 470nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411nm light at 4.5W/m(2) . In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase-3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above-mentioned parameters were not observed when cells were irradiated with 471nm light despite a high irradiance of 4.5W/m(2) , indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411nm (4.5W/m(2) ) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.

BACKGROUND: Endothelial keratoplasty is a promising surgical procedure which may replace penetrating keratoplasty in cases of endothelial cell diseases of the cornea. This method may thereby help to prevent postoperative astigmatism and transplant rejection. METHODS AND RESULTS: A survey of publications reporting about results after endothelial keratoplasty shows that the main problem of this transplantation technique is a postoperative endothelial cell loss which is comparable to or even higher than that observed in penetrating keratoplasty. Improving surgical techniques led to a reduction of the endothelial cell loss, however, cell-based strategies to prevent postoperative cell loss or to enhance the cell densities of donor corneas or endothelial lamellae are rare. DISCUSSION: This review presents an overview of clinical results after endothelial keratoplasty. Current strategies in the field of cell biology and tissue cultivation of corneal endothelial cells, genetic manipulation of the corneal endothelium and tissue engineering strategies aiming at the production of transplantable endothelial cell sheets are described. CONCLUSION: The limited availability of donor corneas makes it mandatory to develop methods in the field of tissue engineering in order to improve corneal endothelial cell survival or to increase corneal endothelial cell density, using interdisciplinary approaches.

We investigated attachment and migration of human retinal pigment epithelial cells (primary, SV40-transfected and ARPE-19) on nanoscopically defined, two-dimensional matrices composed of parallel-aligned collagen type I fibrils. These matrices were used non-cross-linked (native) or after riboflavin/UV-A cross-linking to study cell attachment and migration by time-lapse video microscopy. Expression of collagen type I and IV, MMP-2 and of the collagen-binding integrin subunit α2 were examined by immunofluorescence and Western blotting. SV40-RPE cells quickly attached to the nanostructured collagen matrices and aligned along the collagen fibrils. However, they disrupted both native and cross-linked collagen matrices within 5 h. Primary RPE cells aligned more slowly without destroying either native or cross-linked substrates. Compared to primary RPE cells, ARPE-19 cells showed reduced alignment but partially disrupted the matrices within 20 h after seeding. Expression of the collagen type I-binding integrin subunit α2 was highest in SV40-RPE cells, lower in primary RPE cells and almost undetectable in ARPE-19 cells. Thus, integrin α2 expression levels directly correlated with the degree of cell alignment in all examined RPE cell types. Specific integrin subunit α2-mediated matrix binding was verified by preincubation with an α2-function-blocking antibody, which impaired cell adhesion and alignment to varying degrees in primary and SV40-RPE cells. Since native matrices supported extended and directed primary RPE cell growth, optimizing the matrix production procedure may in the future yield nanostructured collagen matrices serving as transferable cell sheet carriers. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Aim To evaluate the influence of organ culture media on corneal endothelial cell survival. Methods The human corneal endothelial cell line HCEC-12 was cultured in five different media: human corneal endothelial cell (HCEC) growth medium (F99 HCEC ), standard minimal essential corneal organ culture medium (MEM)+2% fetal calf serum (FCS), MEM+5% FCS, and humanised, endothelial serum-free medium (SFM) (with and without antibiotics). A portion of the cells was treated with 0.5 μmol/l staurosporine and examined for signs of apoptosis by assessing mitochondrial membrane polarisation state (intravital JC-1 staining), by YO-PRO-1 and propidium iodide staining, by determining fragmentation of nuclei by sub-G1 DNA content, by immunocytochemistry for cleaved caspase-3, cleaved caspase-8, Bcl2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2), and by western blotting for cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP). Results The number of apoptotic cells in untreated control cultures was significantly higher in MEM compared with F99 HCEC and SFM. Staurosporine treatment induced apoptosis in all tested cultures to varying degrees. Cells cultured in MEM showed stronger staining for cleaved caspase-3, cleaved caspase-8, Bax, Bcl-2 and cleaved PARP, increased sub-G1 DNA content, more propidium iodide- and YO-PRO-1-positive cells, and more mitochondria with depolarised membranes. All parameters were significantly higher in MEM compared with F99 HCEC and SFM. SFM cultures were significantly less susceptible to cell stress. Conclusion SFM is superior to MEM in promoting HCEC survival.

Recently, it was possible to show that human corneal endothelial cells (HCEC) can be cultured on thermo-responsive polymer substrates, and can be harvested as entire cell sheets without losing viability. We sought to study HCEC sheet cultivation on such cell culture carriers under serum-free conditions as the next consequential step in developing methods for generation of corneal endothelial cell transplants.An immortalized heterogenous HCEC population and two immortalized, clonally grown HCEC lines (HCEC-B4G12 and HCEC-H9C1) were cultured on thermo-responsive substrates under serum-supplemented and serum-free culture conditions. Cell sheets were characterized by phase contrast microscopy and by immunofluorescent staining for ZO-1, Na(+),K(+)-ATPase, and vinculin.All tested HCEC populations were able to adhere, spread and proliferate on thermo-responsive substrates under serum-supplemented conditions. Under serum-free conditions, pre-coating of the polymer substrates with ECM proteins was necessary to facilitate attachment and spreading of the cells, except in the case of HCEC-B4G12 cells. The heterogenous HCEC population formed closed monolayers, properly localized ZO-1 to lateral cell borders, and had moderate vinculin levels under serum-free, and higher vinculin levels under serum-supplemented culture conditions. HCEC-B4G12 cells formed closed monolayers, showed proper localization of ZO-1 and Na(+),K(+)-ATPase to lateral cell borders, and had high vinculin levels irrespective of culture conditions. In contrast, HCEC-H9C1 cells had lowest vinculin levels under serum-supplemented, and higher vinculin levels under serum-free culture conditions. ZO-1 was detected throughout the cytoplasm under both culture conditions. These loosely adherent cells were only able to form a closed monolayer under serum-supplemented conditions.Serum-free production of HCEC sheets is possible. The extremely adherent clonal HCEC line B4G12 produced higher vinculin levels than the other two tested HCEC populations, and showed strong adherence to the thermo-responsive, polymeric culture substratum irrespective of culture conditions. This cell line closely resembles terminally differentiated HCEC in vivo, and was found to be particularly suitable for further studies on HCEC cell sheet engineering.

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 ± 14.5 h and that of H9C1 cells 44.05 ± 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase α1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

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.

Gentle harvesting of corneal endothelial cell sheets grown in culture is of interest for the development of cornea replacement strategies. Thin films of a fast responding copolymer of N-isopropylacrylamide (NiPAAm) and diethyleneglycol methacrylate (DEGMA) with a phase transition temperature of 32°C were prepared and evaluated for that purpose. The polymer layers were immobilized onto fluorocarbon substrates using low pressure argon plasma treatment. Cell culture and detachment experiments were performed with L929 mouse fibroblasts and human corneal endothelial cells (HCEC) at standard conditions. The hydrogel-coated supports were found to permit adhesion, spreading, and proliferation of both cell types. Harvesting of cell sheets was achieved upon lowering the temperature to about 30°C. The formation of a closed monolayer as a crucial prerequisite for maintaining ionic pump function in HCEC was proven by ZO-1 immunostainung. Labeling of fibronectin indicated that the vast majority of the extracellular matrix is detached from the hydrogel coatings together with the cell layer. Inspired by this result, the reuse of the hydrogel-coated culture carriers was investigated confirming the suitability of the substrates for repeated cell harvesting. Altogether, the introduced thermoresponsive coating was found advantageous for the efficient generation of HCEC sheets and will be further utilized in transplantation strategies. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res 80A:, 2007

Background . The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells (HCEC), transplantation of these cells may be an alternative therapeutic option. Materials and methods . In this review methods for the in vitro cultivation of HCEC and their transplantation onto the Descemet membrane of donor corneas are described. Results . In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors. Dependent on the culture conditions, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behaviour. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation onto donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation status of the cells in vitro. Highest cell numbers up to 3000 cells/mm 2 were achieved using a SV40-transformed HCEC-cell line. Monolayer integrity could be demonstrated by positive staining for integrins and tight junction proteins, and pump function of the newly established endothelium was proven by perfusion studies. Conclusions . Methods to transplant HCEC onto human denuded corneas have been successfully established to reconstruct human corneas. Recent developments in genetic manipulation of cells and tissue engineering will be of great help in constructing suitable corneas for keratoplasty. Thus corneal endothelial cell transplantation is one of the promising future possibilities to provide corneas of high quality for patients. Furthermore, improvement of the transplantation technique may lead to a method to directly manipulate the diseased endothelium of patients with corneal endothelial dystrophies.

Transplantation of retinal pigment epithelial (RPE) cells is discussed as a possible therapeutic approach for retinal degeneration. Xenogeneic transplantation of human RPE cells in animal models has been studied extensively. Various methods have been used to identify the graft cells, but these methods interfere with cell behaviour so that the monitored physiological post-transplantation course may be influenced. In the present study, we applied a method for an unequivocal identification of the graft cells without interfering cell metabolism or behaviour using in situ hybridisation (ISH) of human specific Alu sequences. Visualisation of the strong extended nuclear signal of Alu sequences was much easier than that of the small nuclear signals of donor-specific sex chromosome probes. With Alu probe, even single graft cells can be identified and their development can be observed in short-term and long-term studies. With this procedure, we could prove that donor cells were injected correctly into the subretinal space by a special injection technique that we developed previously. In combination with immunohistochemistry, donor cells could be clearly discriminated from macrophages, which contained phagocytosed donor cell fragments. Application of these ISH methods for species-specific identification was valuable for follow-up-studies of RPE transplantation.

Retinal pigmented epithelial (RPE) cell integrity is critical to the maintenance of retina functions and RPE cells do not proliferate in adults. The activation of RPE results in cell proliferation which may be associated with proliferative retinopathy and choroidal melanoma. Mitogen-activated protein kinase (MAPK) is believed to be a key participant in the response to mitogenic stimuli. We therefore investigated the involvement of the extracellular signal-regulated protein kinase (ERK) 1 and 2 during the induction of RPE cell proliferation. After foetal calf serum (FCS) stimulation activation of the Ras/Raf/ERK signalling pathway was detected by Western blotting and immunochemistry, with specific anti-phosphosignalling protein antibodies. Pharmacological and antisense (AS) oligonucleotide (ODN) strategies were used to analyse the signalling involved in FCS-induced RPE cell proliferation. Activation of the small G protein Ras and, to a lesser extent of Raf-1, the kinase directly downstream from Ras, was necessary to FCS-induced cell proliferation. MEK1/2 and ERK1/2 were activated during cell proliferation. Inhibition of MEK1/2 with UO 126 completely abolished ERK1/2 activation and reduced cell proliferation by 33-43%. ERK1/2 depletion by an AS ODN approach reduced cell proliferation by 27-33%, confirming the role of ERK1/2 in the FCS stimulation of RPE cells. We also investigated the role of PKA/cAMP, one of the major inhibitory pathways of ERK1/2. PKA blockade did not modify ERK1/2 activation or cell proliferation. In contrast, agents that increased cAMP concentration, abolished RPE proliferation, and MEK/ERK activation. Moreover, inhibition of the cAMP-activated small G protein Rap1, partially reversed the inhibitory effects of cAMP on cell proliferation and MEK/ERK activation. The requirement for Ras and ERK1/2, the lack of ERK1/2 regulation by PKA and the cAMP/Rap1 counter-regulatory pathway for ERK-mediated cell proliferation suggest complex regulation of signalling in RPE cells. These data may have important implications for the development of more selective models for retinal anti-proliferative therapies.

Background/aims In a previous study, we observed a deleterious effect of serum-supplemented Minimal Essential Medium (MEM) on human corneal endothelial cell survival in a cell culture model. Consequently, here we studied the effects of conventional, serum-supplemented MEM and a serum-free medium in combination with two different deswelling substances on cell survival in whole corneas in a mouse model. Methods Murine corneas were cultured for 4 days in MEM+2% fetal calf serum (FCS) or serum-free Human Endothelial-SFM (SFM), both supplemented with either 6% dextran T500 or 7.5% hydroxyethyl starch (HES) 130/0.4. Cells were examined by differential interference contrast microscopy, H&E staining, immunocytochemistry for cleaved caspase-3, Bcl-2, haem oxygenase-1 and immunoblotting for cleaved caspase-3. Results In MEM, endothelial cells were almost completely lost after 4 days and the number of epithelial cells was markedly reduced. The remaining cells showed fragmented nuclei and were positive for cleaved caspase-3 and strongly positive for Bcl-2. Corneas cultured in SFM retained an almost closed layer of endothelial cells. Fewer cells were positive for Bcl-2, and only a few cells were positive for cleaved caspase-3 even under staurosporine administration. HES supplementation was well tolerated by corneal cells over 4 days, while a 4-day supplementation with dextran resulted in the loss of endothelial and epithelial cells. Conclusions Serum-free medium, Human Endothelial-SFM, promoted cell survival during corneal organ culture better than MEM+2% FCS. HES 130/0.4 appeared to be tolerated better by the cells than dextran T500.

We here provide a brief summary of the characteristics of transient receptor potential channels (TRPs) identified in corneal tissue layers and cells. In general, TRPs are nonselective cation channels which are Ca2+ permeable. Most TRPs serve as thermosensitive molecular sensors (thermo-TRPs). Based on their functional importance, the possibilities are described for drug-targeting TRP activity in a clinical setting. TRPs are expressed in various tissues of the eye including both human corneal epithelial and endothelial layers as well as stromal fibroblasts and stromal nerve fibers. TRP vanilloid type 1 (TRPV1) heat receptor, also known as capsaicin receptor, along with TRP melastatin type 8 (TRPM8) cold receptor, which is also known as menthol receptor, are prototypes of the thermo-TRP family. The TRPV1 functional channel is the most investigated TRP channel in these tissues, owing to its contribution to maintaining tissue homeostasis as well as eliciting wound healing responses to injury. Other thermo-TRP family members identified in these tissues are TRPV2, 3 and 4. Finally, there is the TRP ankyrin type 1 (TRPA1) cold receptor. All of these thermo-TRPs can be activated within specific temperature ranges and transduce such inputs into chemical and electrical signals. Although several recent studies have begun to unravel complex roles for thermo-TRPs such as TRPV1 in corneal layers and resident cells, additional studies are needed to further elucidate their roles in health and disease.

Background: To keep the loss of endothelial cell density in donor corneas to a minimum, a storage medium which is adjusted to their nutritional needs is necessary. Different media, used either serum-supplemented or serum-free, are available. The quality of medium- and serum-batches as well as support of endothelial cell viability by the medium are to be tested with a quality assured screening system that allows routine examination. Methods: A screening system was developed which is based on cell-culture tests with the well-established human corneal endothelial cell line HCEC-12, and therefore can be performed without the need for donor corneas. The cells are plated at a defined density in cell-culture dishes, and are cultured for a defined period of time in the test media. Evaluation is carried out by assaying cell count, activity of cell metabolism (resazurin conversion), and determining the number of apoptotic and necrotic cells (combined vital staining with YO-PRO®-1/propidium iodide and subsequent flow cytometry). Results: Human corneal endothelial cells that are cultured in a medium which is adjusted to their nutritional needs achieve higher cell numbers and show a higher metabolic rate. Simultaneously, the percentage of apoptotic and necrotic cells is lower. The screening system developed in this study allows for easy and reliable detection of slightest differences between different media, different processing steps for same media, and different supplements, as well as different serum batches. Conclusions: The differentiated results show that the screening system is sensitive enough to show even minor quality differences. Therefore, it is more suitable than the hitherto commonly used growth assay with primary, mostly porcine, corneal endothelial cells.

The transplantation of retinal pigment epithelial cells (RPE) in patients with age-related macular degeneration is discussed as a future therapy. A cornea bank can serve as a source for cells that can be isolated, cultivated, HLA-typed and cryopreserved for subsequent tissue-compatible transplantation.RPE cells are isolated enzymatically from donor eyes and are cultured in a specially designed growth medium. After multiplication, one part of the culture is cryopreserved; the other part is subcultured for HLA-typing. Completely typed and morphologically sufficiently well-differentiated cell cultures are registered on a donor list (RPE cell bank) and can be provided for cell transplantations with matching HLA type in patients suffering from RPE degenerative diseases.A total of 461 cell cultures have been prepared since 1996; 116 fully typed and well-differentiated cell cultures are stored in our cell bank. Since January 1998 patients who agreed to have an RPE transplantation have been registered on a waiting list. Seven transplantations have already been performed.RPE cells can be stored cryopreserved in a cell bank and can be kept available for transplantation for a prolonged period of time.

Corneal endothelial diseases lead to severe vision impairment, motivating the transplantation of donor corneae or corneal endothelial lamellae, which is, however, impeded by endothelial cell loss during processing. Therefore, one prioritized aim in corneal tissue engineering is the generation of transplantable human corneal endothelial cell (HCEC) layers. Thermo-responsive cell culture carriers are widely used for non-enzymatic harvest of cell sheets. The current study presents a novel thermo-responsive carrier based on simultaneous electron beam immobilization and cross-linking of poly(vinyl methyl ether) (PVME) on polymeric surfaces, which allows one to adjust layer thickness, stiffness, switching amplitude and functionalization with bioactive molecules to meet cell type specific requirements. The efficacy of this approach for HCEC, which require elaborate cell culture conditions and are strongly adherent to the substratum, is demonstrated. The developed method may pave the way to tissue engineering of corneal endothelium and significantly improve therapeutic options.

PURPOSE: Recently, insertion of immuno-modulatory or anti-apoptotic genes into corneal endothelial cells (HCECs) came into focus. Basic FGF-2 occurs in one secreted (low molecular weight, LMW, 18 kD) and four nuclear (high molecular weight, HMW, 22-34 kD) isoforms. HMW isoforms are known differentiation and survival factors, while LMW FGF-2 is a known mitogen. The effect of FGF-2 overexpression of each of the five known isoforms on HCEC cell survival after lentiviral gene transfer in different culture media was investigated. METHODS: Cells were transduced with lentiviral vectors encoding for each of the five FGF-2 isoforms. Transduction efficiency and expression of individual FGF-2 isoforms was assessed by marker gene transfer and western blotting. Primary HCECs were cultured and transduced in four different media previously described for HCEC cultivation or corneal organ cultivation. Cytotoxic effect of virus infection and a possible rescue effect of FGF-2 overexpression were determined by resazurin conversion assay. RESULTS: Transduction with FGF-2 encoding lentiviral vectors resulted in overexpression of the respective isoform in all tested cell populations. Western blotting after total cell lysis proved nuclear localization of transgenic HMW isoforms. Overexpression of HMW FGF-2-especially 34 kD FGF-2-reduced lentiviral cytotoxicity, while overexpression of LMW FGF-2 aggravated viral cytotoxicity. CONCLUSIONS: Cytotoxicity of lentiviral gene transfer in corneal endothelial cells may be reduced by using bicistronic vectors that encode for the target gene and the 34-kD isoform of human FGF-2. Such cotransduction of a survival factor may increase cell survival after gene transfer, thereby improving gene therapeutic approaches.

Water-filtered infrared-A (wIRA) radiation has been described as supportive for tissue regeneration. We sought to investigate in detail the wIRA effect at different temperatures in 3T3 fibroblasts that were treated with glyoxal to induce formation of advanced glycation end products (AGEs). Nonirradiated and nonglyoxal-treated cells served as controls. Experiments were carried out over a range of 37∘–45∘C with exact temperature monitoring to distinguish between temperature and wIRA effects. Metabolic activity was assessed by resazurin assay. Mitochondrial membrane potential was assessed by JC-1 vital staining. Apoptotic changes were determined by vital staining with annexin V and YO-PRO-1 and determination of subG1 DNA content. Temperature had a dominant effect overriding effects exerted by wIRA or glyoxal treatment. The number of apoptotic cells was significantly higher at 45∘C, while the percentage of healthy cells was significantly lower at 45∘C. WIRA irradiation itself or in combination with glyoxal treatment exerted no damaging effects on the fibroblasts at physiological (37∘–40∘C) or higher (42∘–45∘C) temperatures compared to untreated controls. Temperatures of 45∘C, which can occur during inappropriate application of infrared irradiation, damage cells even in the absence of wIRA or glyoxal application.

The injection of cell suspensions or drugs into the subretinal space is a new promising option of vitreoretinal surgery for the treatment of degenerative retinal disorders. We used a manual oil-hydraulic microinjection pump to subretinally inject suspensions of retinal pigment epithelial cells in Royal College of Surgeons rats and in patients suffering from age-related macular degeneration with geographic atrophy. The histological examination of the treated rat eyes showed that cell suspensions could be placed precisely in the subretinal space. Intra- and postoperative outcome of the patients in the clinical trial revealed no retinal complications during 6 months of follow up. We suggest the oil-hydraulic microinjection pump to be a valuable instrument for controlled and precisely dosed atraumatic infusion or aspiration of small volumes of cell suspensions, fluids or drugs in vitreoretinal surgery. [Ophthalmic Surg Lasers 2002;33:340-342]

The aim of our study was to elucidate the role of wavelength and irradiance in blue light retinal damage. We investigated the impact of blue light emitted from light-emitting diode (LED) modules with peaks at either 411nm (half bandwidth 17nm) or 470nm (half bandwidth 25nm) at defined irradiances of 0.6, 1.5 and 4.5W/m(2) for 411nm and 4.5W/m(2) for 470nm on retinal neuronal (R28) cells in vitro. We observed a reduction in metabolic activity and transmembrane potential of mitochondria when cells were irradiated at 411nm at higher irradiances. Furthermore, production of mitochondrial superoxide radicals increased significantly when cells were irradiated with 411nm light at 4.5W/m(2) . In addition, such irradiation caused an activation of the antioxidative glutathion system. Using vital staining, flow cytometry and western blotting, we were able to show that apoptosis only took place when cells were exposed to 411nm blue light at higher irradiances; necrosis was not observed. Enhanced caspase-3 cleavage product levels confirmed that this effect was dependent on light irradiance. Significant alterations of the above-mentioned parameters were not observed when cells were irradiated with 471nm light despite a high irradiance of 4.5W/m(2) , indicating that the cytotoxic effect of blue light is highly dependent on wavelength. The observed phenomena in R28 cells at 411nm (4.5W/m(2) ) point to an apoptosis pathway elicited by direct mitochondrial damage and increased oxidative stress. Thus, light of 411nm should act via impairment of mitochondrial function by compromising the metabolic situation of these retinal neuronal cells.

PURPOSE To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency. METHODS Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99(HCEC), its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay. RESULTS PFV- and HIV-1-based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1-/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM. CONCLUSIONS HIV-1-based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca(2+) transients and induction of these responses through rises in ambient temperature from 25 degrees C to over 40 degrees C. The currents underlying Ca(2+) transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 microM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 degrees C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La(3+)) (100 microM) and ruthenium-red (RuR) (10 microM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 microM) led to a reversible increase in intracellular Ca(2+) indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.

The retinal pigment epithelium (RPE) is a monolayer of cells adjacent to the photoreceptors of the retina. It plays a crucial role in maintaining photoreceptor health and survival. Degeneration or dysfunction of the RPE can lead to photoreceptor degeneration and as a consequence to visual impairment. The most common diseased state of the RPE becomes manifest in age-related macular degeneration, an increasing cause of blindness in the elderly. RPE cells are therefore of great interest to researchers working in the field of tissue engineering and cell transplantation. In fact, studies in animal models have proven that the transplantation of RPE cells can delay the course of photoreceptor degenerative diseases. Although first attempts to transplant RPE cells into the subretinal space in human individuals suffering from age-related macular degeneration were less successful, RPE cell transplantation is still favored as a future therapeutic option, and much work is done to develop and design cell transplants. Cell banking is a prerequisite to have well-differentiated and characterized cells at hand when needed for research purposes, but also for therapeutic approaches. In this chapter the authors will describe methods to isolate, culture and preserve adult human RPE cells for the purpose of RPE cell banking.

Purpose The human corneal endothelium is essential for the physiology and transparency of the cornea. This is sustained by a number of different regulatory mechanisms and response to various stimuli. Exposure to hypotonic challenge and temperature changes may have major impact. This study was undertaken to identify such possible stimuli pathways in human corneal endothelial cells (HCEC). Methods The functional expression of putative temperature- and osmosensing ion channels was investigated by measurements of intracellular free Ca2+ ([Ca2+]i) with fura-2 and automated patch-clamping (microchip technology). Highly selective agents were used to identify TRPV channel subtypes in an immortalized HCEC population (HCEC-SV40) and two subcloned cell lines (H9C1, B4G12). Results The TRPV1 selective agonist capsaicin (20 µM) increased [Ca2+]i and non-selective cation channel outward currents. Cells pre-treated with the antagonist capsazepine (10 µM) did not show any Ca2+ responses. Similar results were obtained with the TRPV4 selective agonist 4α-PDD (5 µM) and the TRP channel blocker ruthenium red (10 µM). In addition, exposure to hypotonic challenge (150 mOsm) led to an increase in [Ca2+]i whereas the isotonic baseline (control) was stable. Furthermore, temperature rises from room temperature to 40-43 °C led to an increase in [Ca2+]i in HCEC-SV40 and H9C1. Conclusion There is functional expression of TRPV channels in HCEC. Therefore, these cells are able to react to temperature rises by activating of TRPV channels. In particular, TRPV1 and TRPV4 may be functionally expressed which are known as heat receptor and osmosensor respectively. These findings may have direct clinical implication (eye banking procedures, keratoplasty).

Mutation or loss of MerTK as well as deficiency of alphavbeta5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca(2+)-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca(2+)-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca(2+)-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca(2+)-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca(2+)-channels could be regulated downstream of both MerTK and alphavbeta5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.

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.

Cultures of normal choroidal melanocytes are useful in vitro models for the study of melanocyte biology. Current protocols involve the supplementation of culture media with serum, toxins, and phorbol esters, the latter being known as tumour-inducing agents. We therefore sought to establish a protocol to cultivate normal human choroidal melanocytes (NHCMs) without these supplements.NHCMs were isolated by dispase II treatment, after isolation of retinal pigment epithelial (RPE) cells, and seeded in serum-free Melanocyte Growth Medium M2 in uncoated T25 cell culture flasks. Purity of the established cultures was proven by immunocytochemistry. Morphology of the cultured cells was evaluated throughout the entire cultivation period. Eventually, cells were cryopreserved in liquid nitrogen.The cultures underwent at least 11.5 +/- 4.5 doublings before they became senescent or culture was deliberately terminated. Mean generation time was 95.0 +/- 27.7 h. After cryopreservation, generation time was markedly increased, but proliferative capacity of the cells was not impaired. Cultured cells showed bipolar to dendritic morphology; sometimes flattened triangular cells were seen in the cultures. Cultured cells lost pigment after initial seeding but displayed continuous melanogenesis. All cultures stained positive for HMB45 antigen and S-100 and negative for RPE-specific cytokeratins 8, 18. Only few cultures contained single cells that were weakly positive for matrix metalloproteinase (MMP)-2 or MMP-9.The cultivation protocol yields pure cultures of NHCMs that can successfully be maintained for several months without the use of serum, tumour-inducing substances, or toxins.

Changes in the phenotype of retinal pigment epithelium (RPE) cells in vitro are associated with medium conditions and changes in function. Main goals in RPE tissue engineering are cell propagation in serum-free defined culture conditions, resulting in cells exhibiting differentiated morphology and functioning in vitro.To compare the effects of various media and supplements on cell function, an optimized high-throughput phagocytosis assay was developed. Adult human SV40-RPE cells were cultured. Test media included: MEM(E), DMEM, F99, SFM and hSFM, with or without supplements. SNAFL-2 labelled OS were added to RPE in vitro for 4 h and phagocytic binding and uptake were measured.RPE phagocytosis was of different magnitude depending on the serum-free basic cell culture media in the following order: hSFM, SFMDMEM, MEMF99. Choroid-conditioned medium (ChCM) decreased phagocytosis dose dependently. Whereas 1% retinal extract (RE) supplementation increased, higher concentrations decreased phagocytosis. Addition of 10% FCS increased phagocytosis. 15% ChCM quenched the stimulation induced by 10% FCS, an effect which could be reversed by the addition of 1% RE.Cell culture media and RPE environmental factors exert substantial and differential alteration of RPE phagocytic ability. Phagocytosis in a serum-free defined medium is superior to unsupplemented basic media, but still differs from serum-supplemented media (F99RPE) designed for cell propagation. We conclude that media SFM or hSFM promoted phagocytosis most, and application of FCS or 1% RE supports phagocytosis. Unknown factors from neighbouring tissues (retina and choroid) affect phagocytosis differently, suggesting a role in retinal pathogenesis. The results will support identification of specific environmental factors and facilitate design of cell culture media.

PURPOSE. Retinal pigment epithelial (RPE) cell death is an important step in the pathogenesis of ocular diseases. JNK1 and P38 kinase, two stress-activated kinases, play key roles relaying stress signals leading to cell death through cyclin D1 and c-Myc. Recently, stress-activated kinases have been shown to regulate cell proliferation. In the current study, the involvement of the JNK1 and P38 kinase signaling pathways in RPE cell proliferation and death was investigated. METHODS. RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the JNK1 and P38 kinase cascades and their potential targets was detected by Western blot analysis. Pharmacologic inhibitors and activators, and antisense oligodeoxynucleotides (ODN) directed against the stress kinases were used to analyze the signaling involved in RPE cell death. RESULTS. P38 and JNK1 and their respective upstream activating kinases, MKK3/6 and -4, were all transiently activated in FCS-stimulated RPE cell cultures. Ras controlled only the activation of JNK1, whereas Rho transmitted the activation of both JNK1 and P38, suggesting parallel signaling pathways and cross talk between the two kinases. Pharmacologic inhibition of JNK1 did not affect cell proliferation in FCS-stimulated cells. Inactivation of P38 kinase and antisense ODN-induced downregulation of P38 kinase also had no affect on cell proliferation. Long-term, high-level activation of JNK1 and P38 kinase occurred during serum depletion-induced RPE cell death. Over-activation of JNK1 and P38 kinase was also observed during pharmacologically induced cell death, suggesting that this process is common to RPE cell-death-signaling pathways induced by various stress stimuli. Cell death mediated by the overactivation of JNK1 and P38 kinase was cyclin Dl- and c-Myc-independent. CONCLUSIONS. The inhibition of JNK1 or P38 kinase had no effect on FCS-stimulated proliferation of RPE cells, whereas the overactivation of these two enzymes was involved in RPE cell death in FCS-depleted cultures. Parallel upstream signaling pathways and cross talk between the two kinases suggest that the regulation of signaling in RPE cell death is complex.

Purpose To use nanoscopically defined, two-dimensional matrices assembled from aligned collagen type I fibrils as a sheet substratum for in vitro cultivation of human corneal endothelial cells (HCECs). To assess the effect of matrix architecture on HCEC morphology and to characterize integrin-mediated HCEC-matrix interaction. Methods Cell alignment and cell-matrix interactions of primary HCECs and three different immortalized HCEC populations on native and UV-cross-linked collagen type I matrices were examined by time-lapse microscopy. Specific integrin α(2)β(1) binding to the collagen matrix was demonstrated using a function-blocking α(2) antibody. Integrin α(2) subunit expression levels of the four HCEC populations were analyzed by Western blot analysis. Results All HCEC populations aligned along the oriented collagen fibrils. Primary HCECs and, to a lesser extent, the other tested HCEC populations exerted high traction forces, leading to progressive matrix destruction. Cross-linking of the collagen matrices considerably increased matrix stability. Integrin subunit α(2) expression levels of the four cell types correlated with the degree of cell alignment and exertion of traction forces. In turn, blocking integrin subunit α(2) reduced cell alignment and prevented matrix destruction. Conclusions HCECs align directionally along parallel arrays of collagen type I fibrils. The interactions of HCECs with collagen type I are primarily mediated by integrin α(2)β(1). Integrin subunit α(2) levels correlate with matrix contraction and subsequent destruction. Sustained cultivation of HCECs on ultrathin collagen matrices thus requires matrix cross-linking and moderate integrin α(2)β(1) expression levels.