Establishment of human retinal microvascular endothelial cells with extended life-span.

AIM: To generate and characterize a telomerase-immortalized human retinal microvascular endothelial cell (HREC) line. This cell line may be utilized as an in vitro model to study the molecular basis of several diseases of the human retina. MATERIALS AND METHODS: Primary retinal neuronal cells were isolated and transfected with plasmid encoding full-length human telomerase reverse transcriptase (hTERT). Transfected cells were selected and characterized to determine telomerase activity, karyotype, proliferative capacity and functionality. RESULTS: HREC-hTERT cells appear morphologically similar to primary endothelial cells and have an extended in vitro life-span. HREC-hTERT cells express the progenitor/stem cell marker nestin. They have active telomerase and a high proliferative capacity. These cells also maintain a diploid karyotype. The HREC-hTERT cells showed high colony-formation capacity and plating efficiency compared to the primary cells. These cells are capable of differentiation into neuronal and glial cell phenotypes and the differentiated cells express the astrocyte marker glial fibrillary acidic protein (GFAP) and the neuronal marker microtubule-associated protein-2 (MAP2), respectively. CONCLUSION: The in vitro life-span of human retinal neuronal endothelial cells can be extended by ectopic expression of hTERT without altering the genetic stability and functionality of these cells. These cells will be a valuable tool to further our understanding on the role of HRECs in the human blood-retinal-barrier and in angiogenesis and neovascularization.

Efficient nanoparticle mediated sustained RNA interference in human primary endothelial cells.

Endothelium forms an important target for drug and/or gene therapy since endothelial cells play critical roles in angiogenesis and vascular functions and are associated with various pathophysiological conditions. RNA mediated gene silencing presents a new therapeutic approach to overcome many such diseases, but the major challenge of such an approach is to ensure minimal toxicity and effective transfection efficiency of short hairpin RNA (shRNA) to primary endothelial cells. In the present study, we formulated shAnnexin A2 loaded poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles which produced intracellular small interfering RNA (siRNA) against Annexin A2 and brought about the downregulation of Annexin A2. The per cent encapsulation of the plasmid within the nanoparticle was found to be 57.65%. We compared our nanoparticle based transfections with Lipofectamine mediated transfection, and our studies show that nanoparticle based transfection efficiency is very high (~97%) and is more sustained compared to conventional Lipofectamine mediated transfections in primary retinal microvascular endothelial cells and human cancer cell lines. Our findings also show that the shAnnexin A2 loaded PLGA nanoparticles had minimal toxicity with almost 95% of cells being viable 24 h post-transfection while Lipofectamine based transfections resulted in only 30% viable cells. Therefore, PLGA nanoparticle based transfection may be used for efficient siRNA transfection to human primary endothelial and cancer cells. This may serve as a potential adjuvant treatment option for diseases such as diabetic retinopathy, retinopathy of prematurity and age related macular degeneration besides various cancers.

Effect of down-regulation of aquaporins in human corneal endothelial and epithelial cell lines.

PURPOSE: The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively. METHODS: AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT-PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay. RESULTS: In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK. CONCLUSIONS: AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.

Presence and distribution of 14-3-3 proteins in human ocular surface tissues.

PURPOSE: 14-3-3 is a highly conserved, ubiquitously expressed family of proteins. At least seven mammalian isoforms (beta, epsilon, gamma, eta, theta, sigma, and zeta) are known. These proteins associate with over 200 different target molecules and activate several downstream signaling cascades involved in the regulation of metabolism, cell cycle, apoptosis, protein trafficking, transcription, stress responses, and malignant transformations. We are interested in the role of these proteins in the mechanisms regulating homeostasis and the pathologies of the human ocular surface. Therefore, our purpose is to determine the expression of the 14-3-3 proteins in the human cornea, the conjunctiva, and the primary cells comprising these tissues. METHODS: Using immunofluorescence, we determined the expression of 14-3-3 beta, epsilon, gamma, eta, theta, sigma, and zeta in paraffin sections of the human cornea and conjunctiva. Using indirect immunofluorescence and western blot analysis, we also determined the expression of these isoforms in primary corneal epithelial cells, keratocytes, endothelial cells, and primary conjunctival epithelial cells. The expressions of these isoforms in primary epithelial and endothelial cells were compared with the same expressions in several corneal cell lines. Western blot analysis was used to determine the presence of 14-3-3 isoforms in the culture medium from corneal epithelial cells, cell lines, and the tear fluid. RESULTS: All the 14-3-3 isoforms were expressed in the corneal and conjunctival epithelia as well as primary epithelial cells and cell lines. Expression of 14-3-3 sigma was confined to epithelial cells and was secreted into the culture medium of primary cells and cell lines. We also report for the first time that two of the secreted isoforms, 14-3-3 gamma and zeta, are also present in the human tear fluid. CONCLUSIONS: We have determined that all the mammalian 14-3-3 isoforms are expressed in the human cornea, conjunctiva, and the component cells and that the 14-3-3 sigma isoform was found to be epithelial cell specific. We propose that the intracellular and extracellular presence of 14-3-3 sigma suggest its involvement in the epithelia specific signaling pathways.