Primary Human Corneal Endothelial Cells Research Articles

Properties of Corneas Reconstructed with Cultured Human Corneal Endothelial Cells and Human Corneal Stroma

To examine the properties of corneas tissue-engineered with cultured human corneal endothelial cells (HCEC) and human corneal stroma. Primary HCEC cultures were established from endothelial cell layer explants and propagated on culture dishes coated with bovine corneal endothelial extracellular matrix. A cell suspension of HCEC at the fifth passage was transferred onto human corneal stroma deprived of endothelial cells, and the corneas were gently centrifuged to enhance cell attachment. The cell density of the tissue-engineered corneas was examined after staining with alizarin red and trypan blue. The tissue-engineered corneas were histologically examined by light and electron microscopy. The pump function of the tissue-engineered corneas was measured using an Ussing chamber. The mean endothelial cell density of four tissue-engineered corneas was 2380 +/- 264 cells/mm(2) (mean +/- SD). HCEC on the tissue-engineered corneas had a morphology similar to HCEC in vivo. The pump function parameters of the tissue-engineered corneas were 55%-75% of those of normal corneas. HCEC on the tissue-engineered corneas have morphology and cellular density similar to HCEC in vivo, whereas the pump function of the tissue-engineered corneas was lower than in normal corneas.

Expression and Activity of Cell Cycle-Regulatory Proteins in Normal and Transformed Corneal Endothelial Cells

Corneal endothelial cells have a limited capacity for proliferation. Upon transformation with the SV40 large T antigen, however, these cells undergo division and grow rapidly. In order to gain insight into the control mechanisms that determine this proliferative switch, we investigated the expression level and activity of various known cell cycle-regulatory proteins in these cells. Primary human and rabbit corneal endothelial cells were transduced in vitro with a replication-defective adenovirus containing SV40 large T antigen, and subsequently the expression and activity of cell cycle-regulatory proteins was analyzed. Cells transduced with large T antigen exhibited strongly increased activity of cyclin-dependent kinases. This increase correlated with the elevated expression of various cyclin-dependent kinase subunits, such as cyclin A, and to a lesser extent, cyclin D, cdk2, and cdk4. Furthermore, the expression of two cyclin-dependent kinase inhibitors, p21(WAF1) and p27(KIP1), which was high in primary human cells (but not in primary rabbit cells), was strongly reduced in large T-antigen transduced cells. Thus, the remarkably low proliferative activity of normal human corneal endothelial cells appears to be regulated at two levels: the expression of certain cell cycle-regulatory proteins that are essential for cell cycle progression is extremely low (cyclin A) or somewhat low (cdk2 and cdk4); but the amount of p21 and p27, inhibitors of cell cycle progression, is very high. As a consequence, the enzymatic activity of cyclin-dependent kinase is below detectable levels. However, the growth-inhibitory status of these components is clearly reversible: upon transduction with large T antigen, the expression of cyclin A, cyclin D, cdk2, and cdk4 is induced, whereas the expression of p21 and p27 is inhibited, and the cells proliferate. Thus, our study provides insight into the molecular basis of the attenuated proliferation of corneal endothelial cells and suggests potential targets that could be manipulated for the purpose of therapeutic interventions aimed at renewed cell growth.

Corneal Endothelial Hyaluronidase

The possible role of the human corneal endothelium in the turnover of anterior chamber hyaluronic acid (HA) was investigated. Hyaluronidase, an endoglycosidase that degrades HA and other glycosaminoglycans, is thought to play a role in HA homeostasis. The presence of hyaluronidase in the corneal endothelium was demonstrated immunohistochemically in sections from normal adult human cornea. Additionally, by using a modified enzyme-linked immunosorbent assay-like assay, active hyaluronidase was detected in the supernatant from primary culture human corneal endothelial cells. The optimal activity for the corneal endothelial hyaluronidase was in the acid range (pH 4.0), similar to previously isolated lysosomal hyaluronidase. Further immunohistochemistry showed that the corneal endothelial cells also express CD44, the receptor for HA, which would allow endocytosis of HA. Human corneal endothelial hyaluronidase may play a role in normal anterior segment HA metabolism and in the degradation of highly concentrated HA used as a visco-elastic.

Transformation of human corneal endothelial cells by microinjection of oncogenes

Human corneal endothelial cells (HCEC) were transfected with some cloned oncogenes. The direct microinjection of either early region (E1) genes of monkey (SA7) and human (Ad5) adenoviruses or Ha-ras oncogen in conjunction with the Ad5 Ela-gene into embryonic HCEC nuclei was shown to result in immortalization of these cells. 3 independent immortalized HCEC lines were established in their growth and morphological properties were studied. These properties were very similar to those of primary HCEC, but unlike primary HCEC the immortalized cells didn't need the endothelial cell growth factor.

Neodymium:YAG laser interaction with intraocular lenses: an in vitro toxicity assay.

Use of the Nd:YAG laser is an effective technique to open an opacified posterior lens capsule. However, in the presence of a posterior chamber intraocular lens (IOL), precise focusing of the laser on the capsule is required to avoid pitting the lens optic. The question has been raised whether toxic products may result from laser damage to the IOL. We addressed this issue in the present study by exposing primary human corneal endothelial cell and human corneal organ cultures to solutions produced by purposefully hitting IOLs immersed in cell growth medium with a Nd:YAG laser. The lenses studied were lathe-cut polymethylmethacrylate (PMMA), injection-molded non-UV PMMA, injection-molded UV PMMA, and cast-molded UV PMMA. Samples of each material were irradiated in a holder containing 1 ml of cell culture medium using the following conditions: 5, 10, and 50 laser bursts at 10 millijoules (mJ), and 50 laser bursts at 5 mJ. The solutions were applied to the endothelial cell cultures (all materials) and to the corneal organ cultures (injection-molded non-UV lenses only). There was no toxicity in either assay for any of the materials studied.