Human Trabecular Cell Cultures Research Articles

The effect of TGF-β2 on human trabecular meshwork extracellular proteolytic system

Our study aimed to investigate whether transforming growth factor-β2 (TGF-β2), increased in the aqueous humor of eyes with primary open angle glaucoma (POAG), can affect factors responsible for the activity of matrix metalloproteinases (MMPs) in human trabecular cell cultures. With this goal in mind cultures of human trabecular meshwork (hTM) cells derived from 8 donors were treated with TGF-β2 for 24, 36 and 48 hr. Influence of TGF-β2 on expression of MMP-2, MMP-9, membrane type 1-MMP (MT1-MMP) and plasminogen activator inhibitor-1 (PAI-1) was examined using RT-PCR, Northern Blot, Western Blot and zymography. The influence of TGF-β2 treatment on PAI-1 expression was also investigated using immunohistochemistry. It appeared that treatment with TGF-β2 significantly increased expression of the proform of MMP-2, whereas the active form was not detectable. MMP-9 and MT1-MMP expression were not influenced by TGF-β2 treatment. There was, however, a significant increase in PAI-1 expression. To investigate whether transformation of the proform of MMP-2 to the active form was inhibited by PAI-1, the influence of treatment with TGF-β2 and a PAI-1 neutralizing antibody on MMP-2 was investigated using zymogram method. With this treatment protocol the active form of MMP-2 was clearly visible, indicating that TGF-β2 enhancement of the PAI-1-expression decreases MMP activity. Inhibition of MMP activity through elevated levels of TGF-β2 might contribute to the increase in ECM in the trabecular meshwork of glaucomatous eyes.

Proliferation and differentiation of human trabecular osteoblastic cells on hydroxyapatite

In order to evaluate whether human osteoblastic cells differentiate normally on hydroxyapatite, we have compared the adhesion, proliferation, and differentiation of human trabecular (HT) osteoblastic cells on synthetic-dense hydroxyapatite and on standard plastic culture. We show here that initial HT cell attachment was 4-fold lower on hydroxyapatite than on plastic after 4 h of culture, and that normal cell attachment on hydroxyapatite was restored after 18 h of culture. HT cell proliferation was similar on the two substrates at 2-8 days of culture, but was lower on hydroxyapatite compared to plastic after 15 and 28 days of culture, as evaluated by DNA synthesis or cell number. HT cells cultured on both substrates produced an abundant extracellular matrix which immunostained for Type I collagen. The levels of carboxyterminal propeptide of Type I procollagen (P1CP) in the medium were lower in HT cell cultures on hydroxyapatite than on plastic. In addition, (3H)-proline incorporation into matrix proteins and the mean thickness of matrix layers were 52% and 26% lower, respectively, on hydroxyapatite compared to plastic after 4 weeks of culture, indicating that the total collagenous matrix synthesized by HT cells was lower on hydroxyapatite. However, (3H)-proline and calcium uptake expressed per cell was higher on hydroxyapatite than on plastic. The results show that human osteoblastic cells attach, proliferate, and differentiate on dense hydroxyapatite with a sequence similar to that of plastic. However, the growth of human osteoblastic cells is lower on hydroxyapatite in long-term culture, which results in a reduced amount of extracellular matrix, although matrix production per cell may be increased.

Dexamethasone Treatment Decreases Hyaluronan-formation by Primate Trabecular Meshwork Cells in Vitro

The functional significance of Hyaluronan (HA) present in the cribriform layer of Schlemm's canal is not known. It may contribute to the actual outflow resistance but the relatively inert molecule might also be necessary to prevent adherence of larger molecules to the cribriform network. Thus HA might rather prevent an increase in outflow resistance. It is well known that treatment with Dexamethasone (DM) in a number of patients leads to an increase in intraocular pressure presumably due to an increase in outflow resistance. To clarify whether an imbalance in HA formation might be involved in these changes we have treated confluent cultures of human trabecular cells as well as control cell lines (ciliary muscle cells, scleral fibroblasts) with 500 nMDM for 24 hr or 12 days and have measured HA-synthesis using incorporation assays with 0.05 mD-[6-3H] Glucosamine hydrochloride. In all six cell lines investigated there was a significant decrease in HA-synthesis following short and long term treatment with DM when compared with the untreated controls. This reaction of trabecular cells to DM treatment is different from the DM effect reported on the synthesis of many other components of the extracellular matrix like fibronectin and elastin which increase after DM treatment. If the DM-effect seen in cell cultures plays a role in vivo decreased formation in HA could result in impaired function of the outflow pathways.

Neural Crest Origin of Human Trabecular Meshwork and Its Implications for the Pathogenesis of Glaucoma

We used an immunohistochemical method to examine the distribution of neuronal-specific enolase in the trabecular meshwork of adult normal human eyes, as well as in primary cultures of human trabecular cells maintained in vitro for up to 44 days with or without the addition of nerve growth factor. In tissue sections of the globes, neuronal-specific enolase was present in the cells of the anterior region of the meshwork and those of the inner uveal beams. The cells of the posterior region of the meshwork stained variably with antiserum against neuronal-specific enolase. Cultured trabecular cells were initially neuronal-specific enolase-positive, but became negative after 18 to 21 days in vitro. The addition of nerve growth factor restored the expression of neuronal-specific enolase in these cultured cells. Because the presence of neuronal-specific enolase in normal cells is believed to indicate their differentiation from neuroectoderm, our results provide evidence that the trabecular cells in human eyes are derived from the embryonic neural crest. Our finding also supports the hypothesis that abnormal migration or a defective terminal induction of the neural crest cells has a role in certain ocular diseases that are characterized by chamber angle anomalies or primary glaucoma.