L-Carnitine Suppresses Transient Receptor Potential Vanilloid Type 1 Activation in Human Corneal Epithelial Cells.

Tear film hyperosmolarity induces dry eye syndrome (DES) through transient receptor potential vanilloid type 1 (TRPV1) activation. L-carnitine is a viable therapeutic agent since it protects against this hypertonicity-induced response. Here, we investigated whether L-carnitine inhibits TRPV1 activation by blocking heat- or capsaicin-induced increases in Ca influx or hyperosmotic stress-induced cell volume shrinkage in a human corneal epithelial cell line (HCE-T).

Induction of vascular endothelial growth factor-A in human retinal and endothelial cells in response to glyoxal.

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.

TRPV4 Stimulation Level Regulates Ca-Dependent Control of Human Corneal Endothelial Cell Viability and Survival.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line.

Effects of human platelet lysate on the growth of cultured human corneal endothelial cells.

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.

L-carnitine suppresses transient receptor potential vanilloid type 1 activity and myofibroblast transdifferentiation in human corneal keratocytes.

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).

Stress responses of human retinal pigment epithelial cells to glyoxal.

Purpose: 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.

Methods: Flow cytometry was combined with either: 1) immunocytochemical staining to detect glyoxal induced formation of N-carboxymethyllysine (CML)-modifications of intracellular proteins (AGEs) and changes in the production of stress response proteins; or 2) vital staining to determine apoptosis rates (annexin V binding), formation of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and changes in intracellular pH upon treatment of cells with glyoxal.

Effects of Narrow-band IR-A and of Water-Filtered Infrared A on Fibroblasts.

Exposures of the skin with electromagnetic radiation of wavelengths between 670 nm and 1400 nm are often used as a general treatment to improve wound healing and reduce pain, for example, in chronic diabetic skin lesions. We investigated the effects of water-filtered infrared A (wIRA) and of narrow-band IR-A provided by a light-emitting diode LED (LED-IR-A) irradiation in vitro on 3T3 fibroblast cultures under defined conditions with and without glyoxal administration. Glyoxal triggers the formation of advanced glycation end products, thereby mimicking a diabetic metabolic state.

Effect of different culture media and deswelling agents on survival of human corneal endothelial and epithelial cells in vitro.

Purpose: To examine the effects of media and deswelling agents on human corneal endothelial and epithelial cell viability using a previously developed screening system.

Methods: The human corneal endothelial cell line HCEC-12 and the human corneal epithelial cell line HCE-T were cultured in four different corneal organ culture media (serum-supplemented: MEM +2 % FCS, CorneaMax®/CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha-2 and -3) with and without 6 % dextran T500 or 7 % HES 130/0.4.

[Effect of autologous platelet concentrates on the anatomical and functional outcome of late stage macular hole surgery: A retrospective analysis].

Background: 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.

Thermo-responsive cell culture carriers based on poly(vinyl methyl ether)-the effect of biomolecular ligands to balance cell adhesion and stimulated detachment.

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(-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.

Sutureless fixation of amniotic membrane for therapy of ocular surface disorders.

Amniotic membrane is applied to the diseased ocular surface to stimulate wound healing and tissue repair, because it releases supportive growth factors and cytokines. These effects fade within about a week after application, necessitating repeated application. Generally, amniotic membrane is fixed with sutures to the ocular surface, but surgical intervention at the inflamed or diseased site can be detrimental.

Temperature-sensitive transient receptor potential channels in corneal tissue layers and cells.

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 Ca(2+) permeable. Most TRPs serve as thermosensitive molecular sensors (thermo-TRPs).

Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas.

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.

Quality assessment of corneal storage media and their components.

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.

Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells.

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.

Tissue engineering of the corneal endothelium: a review of carrier materials.

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.

Effects of temperature and water-filtered infrared-A alone or in combination on healthy and glyoxal-stressed fibroblast cultures.

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.

Overexpression of human HMW FGF-2 but not LMW FGF-2 reduces the cytotoxic effect of lentiviral gene transfer in human corneal endothelial cells.

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.

Characterization of transient receptor potential vanilloid channel 4 (TRPV4) in human corneal endothelial cells.

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.

Blue light stress in retinal neuronal (R28) cells is dependent on wavelength range and irradiance.

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.

Pseudotyping and culture conditions affect efficiency and cytotoxicity of retroviral gene transfer to human corneal endothelial 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).

Retinal pigment epithelium cell alignment on nanostructured collagen matrices.

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 α(2) were examined by immunofluorescence and Western blotting.

Serum-free corneal organ culture medium (SFM) but not conventional minimal essential organ culture medium (MEM) protects human corneal endothelial cells from apoptotic and necrotic cell death.

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.

Alignment and cell-matrix interactions of human corneal endothelial cells on nanostructured collagen type I matrices.

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.