Elisa Toropainen

Endoplasmic Reticulum Stress in Age-Related Macular Degeneration: Trigger for Neovascularization

Age-related macular degeneration (AMD) can be classified into two main categories: the atrophic, dry form and the exudative, wet form. The crucial difference between dry and wet AMD is the development of choroidal neovascularization in wet AMD. One fundamental cause of the neovascularization is the increased expression of VEGF (vascular endothelial growth factor) in retinal pigment epithelial cells. Progression of AMD is linked to augmentation of cellular stress, for example, oxidative stress, proteotoxic stress, inflammation and hypoxia. All these conditions can trigger stress in endoplasmic reticulum (ER), which maintains protein quality control in cells. ER stress induces the unfolded protein response (UPR) via IRE1 (inositol-requiring protein-1), PERK (protein kinase RNA-like ER kinase) and ATF6 (activating transcription factor-6) transducers. UPR signaling is a double-edged sword, that is, it can restore cellular homeostasis as far as possible, but ultimately may lead to chronic, overwhelming stress that can cause apoptotic cell death. Interestingly, ER stress is a well-known inducer of angiogenesis in cancer. Moreover, stress conditions associated with the progress of AMD can induce the expression of VEGF. We discuss the role of ER stress in the regulation of neovascularization and the conversion of dry AMD to its wet, detrimental counterpart.

Paracellular and passive transcellular permeability in immortalized human corneal epithelial cell culture model.

A cell culture model of human corneal epithelium (HCE-model) was recently introduced [Invest. Ophthalmol. Vis. Sci. 42 (2001) 2942] as a tool for ocular drug permeation studies. In this study, passive permeability and esterase activity of the HCE-model were characterised. Immortalised human corneal epithelial cells were grown on collagen coated filters under air-lift. The sensitivity of transcellular permeability to lipophilicity was tested in studies using nine beta-blockers. The size selectivity of the paracellular route was investigated using 16 polyethylene glycol oligomers (PEG). An effusion-like approach was used to estimate porosity and pore sizes of the paracellular space in HCE membrane. Permeability and degradation of fluorescein diacetate to fluorescein in HCE-cells was used to probe the esterase activity of the HCE-model. Drug concentrations were analyzed using HPLC (beta-blockers), LC-MS (PEGs), and fluorometry (fluorescein). Permeabilities were compared to those in the excised rabbit cornea. Penetration of beta-blockers increased with lipophilicity according to a sigmoidal relationship. This was almost similar to the profile in excised cornea. No apical to basolateral directionality was seen in the permeation of beta-blockers. Paracellular permeability of the HCE-model was generally slightly higher than that of the excised rabbit cornea. The HCE-model has larger paracellular pores, but lower pore density than the excised cornea, but the overall paracellular space was fairly similar in both models. The HCE-model shows significant esterase activity (i.e. fluorescein diacetate was converted to free fluorescein). These data on permeability of 27 compounds demonstrate that the barrier of the HCE-model closely resembles that of the excised rabbit cornea. Therefore, the HCE-model is a promising alternative corneal substitute for ocular drug delivery studies.

Simultaneous determination of eight β-blockers by gradient high-performance liquid chromatography with combined ultraviolet and fluorescence detection in corneal permeability studies in vitro

A gradient HPLC method with combined ultraviolet and fluorescence detection was developed for the simultaneous determination of eight beta-blockers (alprenolol, atenolol, metoprolol, nadolol, pindolol, propranolol, sotalol and timolol) in corneal permeability studies in vitro. Fluorescence detection with excitation wavelength at 230 nm and emission at 302 nm was selective for six of the compounds, whereas UV detection at 205 nm was able to detect all the compounds. Calibration was performed with fluorescence detection for six compounds from 50 or 200 nM to 3 microM, and with UV detection for all the eight compounds from 100 or 200 nM to 30 microM. With optimized fluorescence detection, detection limits between 0.7 and 1.3 nM (0.035-0.065 pmol per 50 microl injection) were obtained for atenolol, metoprolol, nadolol and sotalol. A mixture of eight beta-blockers was used in cassette dosing permeability studies with a cultured corneal epithelium. The HPLC method revealed marked differences in the permeation between hydrophilic and lipophilic beta-blockers through the corneal epithelial cell culture model.

Ocular pharmacokinetic modeling using corneal absorption and desorption rates from in vitro permeation experiments with cultured corneal epithelial cells.

AbstractPurpose. To determine corneal absorption and desorption rate constants in a corneal epithelial cell culture model and to apply them to predict ocular pharmacokinetics after topical ocular drug application. Method. In vitro permeation experiments were performed with a mixture of six β-blockers using an immortalized human corneal epithelial cell culture model. Disappearance of the compounds from the apical donor solution and their appearance in the basolateral receiver solution were determined and used to calculate the corneal absorption and desorption rate constants. An ocular pharmacokinetic simulation model was constructed for timolol with the Stella® program using the absorption and desorption rate constants and previously published in vivo pharmacokinetic parameters. Results. The corneal absorption rates of β-blockers increased significantly with the lipophilicity of the compounds. The pharmacokinetic simulation model gave a realistic mean residence time for timolol in the cornea (57 min) and the aqueous humor (90 min). The simulated timolol concentration in the aqueous humor was about 1.8 times higher than the previously published experimental values. Conclusions. The simulation model gave a reasonable estimate of the aqueous humor concentration profile of timolol. This was the first attempt to combine cell culture methods and pharmacokinetic modeling for prediction of ocular pharmacokinetics. The wider applicability of this approach remains to be seen.

Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops

The first objective of the study was to evaluate the transfection of corneal epithelium with non‐viral vectors to secrete transgene products into the tear fluid and aqueous humor. The second goal was to evaluate the differentiated corneal epithelial cell culture for transfection studies.

A critical assessment of in vitro tissue models for ADME and drug delivery.

Cultured cells are widely used in the evaluation of new drugs and drug delivery systems. Cells can be grown at different levels of complexity ranging from simple reductionist models to complex organotypic models. The models are based on primary, secondary or stem cell derived cell cultures. Generation of tissue mimics with cultured cells is a difficult task, because the tissues have well-defined morphology, complex protein expression patterns and multiple inter-linked functions. Development of organotypic cell culture models requires proper biomaterial matrix and cell culture protocols that are able to guide the cells to the correct phenotype. This review illustrates the critical features of the cell culture models and, then, selected models are discussed in more detail (epidermal, corneal epithelial, retinal pigment epithelium, and hepatocyte models). The cell models are critically evaluated paying attention to the level of characterization and reliability of in vivo translation. Properties of the cell models must be characterized in detail using multiple biological assays and broad sets of model drugs. Robust in vivo predictions can be achieved with well-characterized cell models that are used in combination with computational methods that will bridge the gap between in vitro cell experiments and physiological situation in vivo in the body.

Liquid chromatographic-electrospray ionization mass spectrometric analysis of neutral and charged polyethylene glycols.

Poly(ethylene glycols) (PEGs) are widely used water soluble and biocompatible polymers. PEGs are suitable as paracellular probes in biomembrane permeability studies because they are hydrophilic and various oligomers have defined molecular sizes. In previous studies corneal and conjunctival permeability for neutral PEGs has been measured, and the results were used to calculate the number and size of the cellular pores. In this study we have developed a high-performance liquid chromatographic-electrospray ionization-mass spectrometric method for analysis of neutral PEGs and positively changed amino PEGs simultaneously. The new method is fast, accurate, sensitive and specific for high throughput analysis. The method was used to evaluate the paracellular permeability of PEGs through a corneal epithelial cell culture. Paracellular pores are negatively charged and it was in our interest to characterize the interactions of positive charge and size of the molecules with the paracellular pores.

Human corneal cell culture models for drug toxicity studies.

In vivo toxicity and absorption studies of topical ocular drugs are problematic, because these studies involve invasive tissue sampling and toxic effects in animal models. Therefore, different human corneal models ranging from simple monolayer cultures to three-dimensional models have been developed for toxicological prediction with in vitro models. Each system has its own set of advantages and disadvantages. Use of non-corneal cells, inadequate characterization of gene-expression profiles, and accumulation of genomic aberrations in human corneal models are typical drawbacks that decrease their reliability and predictive power. In the future, further improvements are needed for verifying comparable expression profiles and cellular properties of human corneal models with their in vivo counterparts. A rapidly expanding stem cell technology combined with tissue engineering may give future opportunities to develop new tools in drug toxicity studies. One approach may be the production of artificial miniature corneas. In addition, there is also a need to use large-scale profiling approaches such as genomics, transcriptomics, proteomics, and metabolomics for understanding of the ocular toxicity.

Loss of NRF-2 and PGC-1α genes leads to retinal pigment epithelium damage resembling dry age-related macular degeneration

Age-related macular degeneration (AMD) is a multi-factorial disease that is the leading cause of irreversible and severe vision loss in the developed countries. It has been suggested that the pathogenesis of dry AMD involves impaired protein degradation in retinal pigment epithelial cells (RPE). RPE cells are constantly exposed to oxidative stress that may lead to the accumulation of damaged cellular proteins, DNA and lipids and evoke tissue deterioration during the aging process. The ubiquitin-proteasome pathway and the lysosomal/autophagosomal pathway are the two major proteolytic systems in eukaryotic cells. NRF-2 (nuclear factor-erythroid 2-related factor-2) and PGC-1α (peroxisome proliferator-activated receptor gamma coactivator-1 alpha) are master transcription factors in the regulation of cellular detoxification. We investigated the role of NRF-2 and PGC-1α in the regulation of RPE cell structure and function by using global double knockout (dKO) mice. The NRF-2/PGC-1α dKO mice exhibited significant age-dependent RPE degeneration, accumulation of the oxidative stress marker, 4-HNE (4-hydroxynonenal), the endoplasmic reticulum stress markers GRP78 (glucose-regulated protein 78) and ATF4 (activating transcription factor 4), and damaged mitochondria. Moreover, levels of protein ubiquitination and autophagy markers p62/SQSTM1 (sequestosome 1), Beclin-1 and LC3B (microtubule associated protein 1 light chain 3 beta) were significantly increased together with the Iba-1 (ionized calcium binding adaptor molecule 1) mononuclear phagocyte marker and an enlargement of RPE size. These histopathological changes of RPE were accompanied by photoreceptor dysmorphology and vision loss as revealed by electroretinography. Consequently, these novel findings suggest that the NRF-2/PGC-1α dKO mouse is a valuable model for investigating the role of proteasomal and autophagy clearance in the RPE and in the development of dry AMD.

Corneal and conjunctival drug permeability: Systematic comparison and pharmacokinetic impact in the eye

ABSTRACT On the surface of the eye, both the cornea and conjunctiva are restricting ocular absorption of topically applied drugs, but barrier contributions of these two membranes have not been systemically compared. Herein, we studied permeability of 32 small molecular drug compounds across an isolated porcine cornea and built a quantitative structure‐property relationship (QSPR) model for the permeability. Corneal drug permeability (data obtained for 25 drug molecules) showed a 52‐fold range in permeability (0.09–4.70×10−6cm/s) and the most important molecular descriptors in predicting the permeability were hydrogen bond donor, polar surface area and halogen ratio. Corneal permeability values were compared to their conjunctival drug permeability values. Ocular drug bioavailability and systemic absorption via conjunctiva were predicted for this drug set with pharmacokinetic calculations. Drug bioavailability in the aqueous humour was simulated to be <5% and trans‐conjunctival systemic absorption was 34–79% of the dose. Loss of drug across the conjunctiva to the blood circulation restricts significantly ocular drug bioavailability and, therefore, ocular absorption does not increase proportionally with the increasing corneal drug permeability.