Insulin growth factor promotes human corneal fibroblast network formation in vitro.

PURPOSE: Corneal fibroblast cell (CFC) reticulation is involved in the structural development of corneal stroma and in wound healing. In an earlier paper, it was reported that the expression of VEGFR-1 by CFCs is related to their reticulogenic properties in vitro and decreases with the age of the donors. The present study was focused on the nonreticulogenic corneal fibroblast population and explored whether these cells can be induced to form cell networks in vitro. METHODS: The network formation was analyzed using an array of signaling pathway inhibitors: wortmannin for PI3 kinase, U0126 for MEK-1/2 kinase, Rottlerin for PKC, farnesyl transferase inhibitor (FTI-277) for Ras, and picropodophyllin (PPP) for IGFR-1. Among the several growth factors studied, IGF seemed to be crucial to cell network formation. The presence of IGF signaling was demonstrated using gene array analysis and was confirmed by RT-PCR and immunocytochemistry and by cell network formation on reduced synthetic basement membrane arrays. The pleiotropic effect of IGF-1 on the cells was analyzed by protein cytokine array. RESULTS: The genesis of reticulation was found to occur via MEK-1/2 and IGFR pathways, since inhibitors of these signaling pathways reduced or prevented cell network formation. The addition of exogenous IGF-1 generated a cell network structure in corneal fibroblasts obtained from healthy donors, indicating the involvement of IGF-1. CONCLUSIONS: IGF signaling and the MEK-1/2 pathway are involved in the cell network formation of corneal fibroblast cells from aged donors.

Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age.

PURPOSE: Mechanisms by which fibroblast networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial growth factor receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from 19 to 89 years. METHODS: Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT-PCR] and VEGFR siRNA transfections), and cell culture. RESULTS: Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel or not (18%). By RT-PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro. CONCLUSIONS: The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals.

Aldosterone modifies hemostasis via upregulation of the protein-C receptor in human vascular endothelium.

In human bone marrow endothelial cell (HBMEC) exposed for 8 h to aldosterone, the microarray screening revealed an upregulation of the mRNAs for six genes and downregulation of mRNAs for four genes, all implicated in hemostasis. In HBMEC, immunocytochemistry revealed the presence of the membrane-bound endothelial protein C receptor (EPCR) whereas the mineralocorticoid receptor (MCR) was present as a nucleo-cytoplasmic. In HBMEC treated with aldosterone the induction of EPCR protein was evident by both FACS analysis and dot blot procedure. When aldosterone-treated HBMEC were incubated with the activated protein C (APC), the partial thromboplastin clotting time (aPTT) increased 2.5-fold over control, from 10 to 25 s. The MCR antagonists aldactone and eplerenone reduced the basal coagulation time in untreated cells to 33.5% and 42% of the control, respectively. These data add an entirely new dimension to delineating the receptor-mediated action of mineralocorticoid hormones.