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Monika Valtink

Publications and source records attributed to Monika Valtink.

9 recordsLinked to original sources
PubMed2007

Thermo-responsive poly(NiPAAm-co-DEGMA) substrates for gentle harvest of human corneal endothelial cell sheets.

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 degrees 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 degrees 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.

Animals↗
PubMed2006

Cell culture conditions affect RPE phagocytic function.

BACKGROUND: 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. METHODS: 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. RESULTS: RPE phagocytosis was of different magnitude depending on the serum-free basic cell culture media in the following order: hSFM, SFM > DMEM, MEM > F99. 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. CONCLUSIONS: 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.

Adult↗
PubMed2004

Prospects for endothelial transplantation.

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/mm2 were achieved using a SV40-transformed HCEC-cell line. Monolayer integrity could be demonstrated by positive staining for integrins and light 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.

Cell Culture Techniques↗
PubMed2003

Experimental rat model for therapeutic retinal pigment epithelium transplantation--unequivocal microscopic identification of human donor cells by in situ hybridisation of human-specific Alu sequences.

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.

Alu Elements↗
PubMed2003

Activation and role of MAP kinase-dependent pathways in retinal pigment epithelium cells: JNK1, P38 kinase, and cell death.

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. Overactivation 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 D1- 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.

Apoptosis↗
PubMed2002

cAMP inhibits the proliferation of retinal pigmented epithelial cells through the inhibition of ERK1/2 in a PKA-independent manner.

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.

Animals↗
PubMed2002

Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation.

PURPOSE: Retinal pigment epithelial (RPE) cell proliferation plays a key role in the pathogenesis of ocular diseases involving the posterior segment. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell proliferation. This study was conducted to investigate the involvement of the extracellular signal-regulated kinase (ERK) pathway, the major MAPK pathway implicated in cell growth during the induction of RPE cell proliferation. METHODS: RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the Ras/Raf/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot analysis and immunochemistry with specific anti-phosphosignaling protein antibodies. Pharmacologic and antisense (AS) oligonucleotide (ODN) strategies were used to analyze the ERK signaling involved in serum-induced cell proliferation. RESULTS: FCS (10%) induced more vigorous RPE cell proliferation than did FGF2, VEGF, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), alone or in combination. Pharmacologic inhibition of Ras and Raf-1 reduced cell proliferation by 67% to 100% and by 62% to 79%, respectively, demonstrating that activation of the Ras/Raf-1 pathway was essential for FCS-induced RPE cell proliferation. MEK1/2, ERK2, and P90 ribosomal S6 kinase (P90(RSK)), the kinases downstream from ERK2, were strongly activated during cell proliferation. Pharmacologic inhibition of MEK1/2 abolished activation of ERK2, but reduced cell proliferation by only 32%, showing that MEK/ERK participates in the signaling involved in RPE cell growth. Both inhibition of ERK2 activation, which reduced cyclin D1 production, and inhibition of cyclin D1 by AS ODN decreased cell proliferation, suggesting that RPE cell proliferation is mediated by cyclin D1 through ERK2. CONCLUSIONS: The requirement for Ras and the regulatory role of ERK2 in cyclin D1 production and in cell proliferation suggest that the Ras/Raf/MEK/ERK pathway plays a key role in the control of RPE cell proliferation. These data may have important implications for the development of more selective methods for the inhibition of retinal proliferation.

Blotting, Western↗
PubMedDate not supplied

Use of an oil-hydraulic microinjection pump for subretinal infusions.

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.

Animals↗