| 1. |
- Björkblom, Benny, et al.
(författare)
-
Xenobiotic- and Serum-Free Culture of Oral Mucosal Epithelial Cells on Contact Lenses
- 2016
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 41:1, s. 20-27
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose/aim: Cultured autologous oral mucosal epithelial cells (OMECs) have proven useful in the treatment of ocular surface disorders. This study is the first to investigate the potential of expanding OMEC in a xenobiotic- and serum-free medium using therapeutic contact lenses (CLs) as a substrate and carrier.Materials and methods: Porcine OMEC were seeded on laminin-coated lotrafilcon A therapeutic CLs with the density of 8x10(4)cells/lens and cultured in a defined serum and xenobiotic-free medium. Confocal immunofluorescence microscopy was used to analyze the following: (1) cellular morphology by using rhodamine-phalloidin staining of F-actin, (2) phenotype by applying antibodies against the progenitor cell marker p63 and the putative stem cell marker ABCG2 and (3) cell viability by using propidium iodide and Hoechst 33342 dual staining.Results: Porcine OMEC attached well to the CLs, and cell-to-cell contacts were evident. After three days in culture, the OMEC displayed a confluent monolayer with uniform cobblestone morphology, whereas stratified cultures with 2-3 layers were formed after six days. No significant difference in expression of p63 was observed after three-day culture (79.414.8%) compared with six-day culture (60.3 +/- 18.9%). ABCG2 expression in the basal cell layer was 6.3 +/- 1.0% and 4.8 +/- 1.8% after three- and six-day culture, respectively. The basal layer viability of cultured OMECs was 99.3 +/- 0.2% and 82.8 +/- 1.1% after three and six days culture, respectively.Conclusions: The use of therapeutic CLs has potential as a substrate and carrier for OMEC cultured in a xenobiotic- and serum-free culture system.
|
|
| 2. |
|
|
| 3. |
|
|
| 4. |
|
|
| 5. |
- Harun-Or-Rashid, Mohammad, 1980-, et al.
(författare)
-
Alpha 2-Adrenergic Receptor Agonist Brimonidine Stimulates ERK1/2 and AKT Signaling via Transactivation of EGF Receptors in the Human MIO-M1 Müller Cell Line
- 2019
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 44:1, s. 34-45
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose: Alpha 2-adrenergic receptor (α2-ADR) agonists are used clinically for a range of indications including reducing elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Animal experiments show that α2-ADR agonists attenuate the injury-induced Müller cell dedifferentiation by a mechanism that involves activation and regulation of extracellular signal-regulated kinase (ERK) 1/2 leading to transactivation of epidermal growth factor receptors (EGFRs). The purpose of this study was to study and corroborate the activation of this system in human cells.Material and Methods: The human Müller cell line MIO-M1 was treated with the α2A-ADR agonist brimonidine in combination with inhibitors for Src-kinase, EGFR-kinase, matrix metalloproteinase (MMP) as well as small interfering RNAs (siRNAs) for the EGFR. The cells were analyzed using immunocytochemistry, quantitative PCR and western blot techniques.Results: Our results show that human MIO-M1 cells express α2A-ADRs and that stimulation of these receptors caused a robust increase of ERK1/2 and protein kinase B (PKB/AKT) (Thr-308) phosphorylation in MIO-M1 cells. P-ERK1/2 and P-AKT (Thr-308) signaling was mediated by Src-kinase and associated with phosphorylation of tyrosine residue of epidermal growth factor receptor (P-EGFR Y1173). In addition, the agonist caused activation of MMPs. These effects could be blocked by Src-kinase inhibitors (PP1, PP2), EGFR-kinase inhibitor (AG1478), EGFR-siRNA and a MMP inhibitor (GM6001).Conclusion: The results confirm that this human Müller cell line responds to ADR stimulation with phosphorylation of ERK and AKT, which suggests that it is possible to pharmacologically target ADR to modulate the early events in human Müller cell dedifferentiation in a similar fashion as been shown for chicken Müller cells.Abbreviations: CRALBP: cellular retinaldehyde binding protein; EGFR: epidermal growth factor receptor; ERK1/2: extracellular signal-regulated kinase 1/2; GS: glutamine synthetase; GPCR: G protein-coupled receptor; IR: immunoreactivity; MAPK: mitogen-activated protein kinase; MMP: matrix metalloproteinase; P-ERK1/2: phospho-ERK1/2; qRT-PCR: quantitative reverse transcriptase PCR
|
|
| 6. |
- Koulikovska, Marina, 1970-, et al.
(författare)
-
Platelet Rich Plasma Prolongs Myofibroblast Accumulation in Corneal Stroma with Incisional Wound
- 2015
-
Ingår i: Current Eye Research. - : Taylor & Francis. - 0271-3683 .- 1460-2202. ; 40:11, s. 1102-1110
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose: The purpose of this study was to determine whether platelet rich plasma (PRP) has an effect on corneal stromal cells in a rat model of wound healing following corneal incision. Materials and Methods: The effect of PRP on corneal wound healing in vivo was investigated in a corneal incision wound model in rats. 40 rats were wounded by deep corneal incision, and treated with either topically administered PRP (20 rats) or sodium chloride (20 rats). At 4 hours and 1, 3, and 5 days after incision, α-smooth muscle actin (α SMA), SMAD2 and SMAD3 expression and apoptosis in stromal cells were evaluated by immunohistochemistry, and IL-1β mRNA expression was evaluated by real time PCR.Results: PRP treated corneas exhibited reduced stromal cell apoptosis at day 3 and day 5 (p = 0.038, and <0.001, respectively) relative to controls. Interleukin-1β mRNA expression, however, was unchanged in PRP treated corneas relative to controls. Topical PRP treatment resulted in a higher proportion of αSMA-positive myofibroblasts recruited to the wound site relative to control corneas. PRP did not affect activation of SMAD2 but activation of SMAD3 was significantly reduced at day 1 (p=0.001) and dramatically increased at day 5 (p=0.032).Conclusions: PRP treatment resulted in suppressed stromal cell apoptosis followed by SMAD3 activation and a greater proportion of myofibroblasts present at the wound site. Suppression of stromal cell apoptosis after corneal wounding by use of a growth factor rich formulation may lead to myofibroblast accumulation by modulation of the TGF-β pathway.
|
|
| 7. |
- Roux, Sandrine Le, et al.
(författare)
-
Transforming Growth Factor Beta 1 Modulates the Functional Expression of the Neurokinin-1 Receptor in Human Keratocytes
- 2016
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 41:8, s. 1035-1043
-
Tidskriftsartikel (refereegranskat)abstract
- PURPOSE: Transforming growth factor beta 1 (TGF-β1) is a cytokine involved in a variety of processes, such as differentiation of fibroblasts into myofibroblasts. TGF-β1 has also been shown to delay the internalization of the neurokinin-1 receptor (NK-1 R) after its activation by its ligand, the neuropeptide substance P (SP). NK-1 R comprises two naturally occurring variants, a full-length and a truncated form, triggering different cellular responses. SP has been shown to affect important events in the cornea - such as stimulating epithelial cell proliferation - processes that are involved in corneal wound healing and thus in maintaining the transparency of the corneal stroma. An impaired signaling through NK-1 R could thus impact the visual quality. We hypothesize that TGF-β1 modulates the expression pattern of NK-1 R in human corneal stroma cells, keratocytes. The purpose of this study was to test that hypothesis.METHODS: Cultures of primary keratocytes were set up with cells derived from healthy human corneas, obtained from donated transplantation graft leftovers, and characterized by immunocytochemistry and Western blot. Immunocytochemistry for TGF-β receptors and NK-1 R was performed. Gene expression was assessed with real-time polymerase chain reaction (qPCR).RESULTS: Expression of TGF-β receptors was confirmed in keratocytes in vitro. Treating the cells with TGF-β1 significantly reduced the gene expression of NK-1 R. Furthermore, immunocytochemistry for NK-1 R demonstrated that it is specifically the expression of the full-length isotype of the receptor that is reduced after treatment with TGF-β1, which was also confirmed with qPCR using a specific probe for the full-length receptor.CONCLUSIONS: TGF-β1 down-regulates the gene expression of the full-length variant of NK-1 R in human keratocytes, which might impact its signaling pathway and thus explain the known delay in internalization after activation by SP seen with TGF-β1 treatment.
|
|
| 8. |
- Skiljic, Dragana, 1985, et al.
(författare)
-
Effects of 17β-Estradiol on Activity, Gene and Protein Expression of Superoxide Dismutases in Primary Cultured Human Lens Epithelial Cells
- 2018
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 1460-2202 .- 0271-3683. ; 43:5, s. 639-646
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose: Protective effects of estradiol against H 2 O 2 -induced oxidative stress have been demonstrated in lens epithelial cells. The purpose of this study was to investigate the effects of 17β-estradiol (E2) on the different superoxide dismutase (SOD) isoenzymes, SOD-1, SOD-2, and SOD-3, as well as estrogen receptors (ERs), ERα and ERβ, in primary cultured human lens epithelial cells (HLECs). Materia ls and methods: HLECs were exposed to 0.1 µM or 1 µM E2 for 1.5 h and 24 h after which the effects were studied. Protein expression and immunolocalization of SOD-1, SOD-2, ERα, and ERβ were studied with Western blot and immunocytochemistry. Total SOD activity was measured, and gene expression analyses were performed for SOD1, SOD2, and SOD3. Results: Increased SOD activity was seen after 1.5 h exposure to both 0.1 µM and 1 µM E2. There were no significant changes in protein or gene expression of the different SODs. Immunolabeling of SOD-1 was evident in the cytosol and nucleus; whereas, SOD-2 was localized in the mitochondria. Both ERα and ERβ were immunolocalized to the nucleus, and mitochondrial localization of ERβ was evident by colocalization with MitoTracker. Both ERα and ERβ showed altered protein expression levels after exposure to E2. Conclusions: The observed increase in SOD activity after exposure to E2 without accompanying increase in gene or protein expression supports a role for E2 in protection against oxidative stress mediated through non-genomic mechanisms.
|
|
| 9. |
- Spinozzi, Daniele, et al.
(författare)
-
Evaluation of the Suitability of Biocompatible Carriers as Artificial Transplants Using Cultured Porcine Corneal Endothelial Cells
- 2019
-
Ingår i: Current Eye Research. - : TAYLOR & FRANCIS INC. - 0271-3683 .- 1460-2202. ; 44:3, s. 243-249
-
Tidskriftsartikel (refereegranskat)abstract
- Purpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK). Materials and Methods: pCECs were isolated, cultured up to P2 and seeded onto LinkCell (TM) bioengineered matrices of 20 mu m (LK20) or 100 mu m (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM). Results: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 +/- 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a "taco" for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma. Conclusions: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.
|
|
| 10. |
- Taylor, Linnéa, et al.
(författare)
-
First Responders: Dynamics of Pre-Gliotic Müller Cell Responses in The Isolated Adult Rat Retina.
- 2015
-
Ingår i: Current Eye Research. - : Informa UK Limited. - 0271-3683 .- 1460-2202. ; 40:12, s. 1245-1260
-
Tidskriftsartikel (refereegranskat)abstract
- Abstract Purpose: To explore the early reactions of the retinal Müller glia in response to retinal insult prior to gliotic remodeling and the sustained upregulation of intermediate filament glial fibrillary acidic protein (GFAP), which has traditionally been considered the most sensitive early indicator of reactive gliosis. Methods: To study pre-gliotic events, we used a model of adult rat retinal explants and related the dynamic expression of GFAP as well as apoptosis, to four key regulators of retinal homeostasis (glutamine synthetase (GS), cellular retinaldehyde binding protein (CRALBP), basic fibroblast growth factor (bFGF), carbonic anhydrase II (CAII)) using immunohistochemistry. Results: We found that a sustained GFAP upregulation couple with gliotic remodeling occurred comparatively late and that this phenomenon was preceded by an initial upregulation followed by depletion of GS, CRALBP, bFGF and CAII in retinal Müller cells. The initial increase of the regulatory proteins, seen after 1-12 h, preceded a first phase of moderate apoptosis, and their depletion after 48 h was followed by massive apoptosis and widespread GFAP upregulation in the Müller cells at 5 days. Conclusion: We conclude that, in the explant model, changes in the expression of the four homeostatic regulatory proteins as well as apoptotic cell death precedes sustained GFAP upregulation and reactive gliosis. Müller cell reactivity has been linked to several retinal conditions, and the herein provided novel information on the dynamics of pre-gliotic events in the lesioned retina may help us understand important pathological mechanisms crucial for future therapeutic intervention.
|
|
