| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 From the Research Division and 2 Beetham Eye Institute, Joslin Diabetes Center, Boston, Massachusetts; and the 3 Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
PURPOSE. Hepatocyte growth factor (HGF), also called scatter factor, stimulates growth and motility in nonocular endothelial cells and smooth muscle cells through its receptor c-Met. Recent reports suggest that HGF is increased in the serum and vitreous of patients with proliferative diabetic retinopathy and that smooth muscle cells and retinal pigment epithelial cells secrete HGF in the eye. However, little is known about HGF’s action in the retina. In this study, the activity, expression, and signaling pathways of HGF were investigated in bovine retinal microvascular endothelial cells (BRECs).
METHODS. Mitogenic and motogeneic effects of HGF on BRECs were examined using cell counts, thymidine uptake, and migration assays. MAP kinase (MAPK) phosphorylation was examined by Western blot analysis. Protein kinase C (PKC), MAPK, and PI3 kinase involvement were evaluated using selective inhibitors and activity assays. Expression of HGF and c-Met was evaluated by reverse transcription–polymerase chain reaction.
RESULTS. HGF and c-Met were both expressed in BRECs. HGF stimulated BREC growth in a time- and dose-dependent manner, observed at HGF concentrations of 5 ng/ml or more and maximal (410%) at 100 ng/ml (P < 0.001). HGF increased BREC migration in a dose-dependent manner with a maximal 3.4-fold increase at 50 ng/ml after 5 hours. HGF induced time- and dose-dependent MAPK phosphorylation, initially evident at 5 minutes (P < 0.001) or 5 ng/ml (P < 0.050) and maximal after 15 minutes (>80-fold, P < 0.001) or 50 ng/ml (>20-fold, P < 0.001), respectively. MAPK phosphorylation was maintained for more than 2 hours. This response was inhibited 31% by 0.1 µm wortmannin and 76% by 30 µm LY294002, another PI3 kinase inhibitor. The non–isoform-selective PKC inhibitor GFX inhibited HGF-induced MAPK phosphorylation by only 15% at 5 µm. Combined PKC and PI3 kinase inhibition was additive (P < 0.05). Cell migration was inhibited 30% by wortmannin (P < 0.01) and 32% by GFX (P < 0.05), and again the effect was additive (P < 0.001). HGF-induced BREC growth was suppressed by PI3 kinase, PKC, or MAPK inhibition (all P < 0.01). HGF (50 ng/ml) stimulated PI3 kinase activity 347% (P < 0.001) and PKC activity 37% (P < 0.05). HGF-induced MAPK phosphorylation and mitogenesis were not inhibited by vascular endothelial growth factor (VEGF)–neutralizing antibody.
CONCLUSIONS. HGF and its receptor are expressed in BREC, and HGF stimulates both BREC growth and migration at concentrations observed in the human eye with diabetic retinopathy. HGF signaling appears to involve activation of both PKC and PI3 kinase, inducing MAPK phosphorylation that is critical for migration and growth. However, VEGF does not appear to mediate these initial HGF effects. These results indicate that HGF could have a significant role in mediating retinal endothelial cell proliferation and migration in diabetic retinopathy, and they begin to elucidate the signal transduction pathway by which this action may occur.
This article has been cited by other articles:
|
|
 |
|
  A. C. Clermont, M. Cahill, H. Salti, S. L. Rook, C. Rask-Madsen, L. Goddard, J. S. Wong, D. Bursell, S. E. Bursell, and L. P. Aiello Hepatocyte Growth Factor Induces Retinal Vascular Permeability via MAP-Kinase and PI-3 Kinase without Altering Retinal Hemodynamics. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2701 - 2708. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Iwabu, K. Smith, F. D. Allen, D. A. Lauffenburger, and A. Wells Epidermal Growth Factor Induces Fibroblast Contractility and Motility via a Protein Kinase C {delta}-dependent Pathway J. Biol. Chem., April 9, 2004; 279(15): 14551 - 14560. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Merkulova-Rainon, P. England, S. Ding, C. Demerens, and G. Tobelem The N-terminal Domain of Hepatocyte Growth Factor Inhibits the Angiogenic Behavior of Endothelial Cells Independently from Binding to the c-met Receptor J. Biol. Chem., September 26, 2003; 278(39): 37400 - 37408. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Ding, T. Merkulova-Rainon, Z. C. Han, and G. Tobelem HGF receptor up-regulation contributes to the angiogenic phenotype of human endothelial cells and promotes angiogenesis in vitro Blood, June 15, 2003; 101(12): 4816 - 4822. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. A. McBain, J. V. Forrester, and C. D. McCaig HGF, MAPK, and a Small Physiological Electric Field Interact during Corneal Epithelial Cell Migration Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 540 - 547. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Reisinger, R. Kaufmann, and J. Gille Increased Sp1 phosphorylation as a mechanism of hepatocyte growth factor (HGF/SF)-induced vascular endothelial growth factor (VEGF/VPF) transcription J. Cell Sci., January 15, 2003; 116(2): 225 - 238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. K. L. Woodward, S. R. Elshaw, A. K. Murray, C. E. Nichols, N. Cross, D. Laws, I. G. Rennie, and K. Sisley Stimulation and Inhibition of Uveal Melanoma Invasion by HGF, GRO, IL-1{alpha} and TGF-{beta} Invest. Ophthalmol. Vis. Sci., October 1, 2002; 43(10): 3144 - 3152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. J. Duh, H. S. Yang, I. Suzuma, M. Miyagi, E. Youngman, K. Mori, M. Katai, L. Yan, K. Suzuma, K. West, et al. Pigment Epithelium-Derived Factor Suppresses Ischemia-Induced Retinal Neovascularization and VEGF-Induced Migration and Growth Invest. Ophthalmol. Vis. Sci., March 1, 2002; 43(3): 821 - 829. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zubilewicz, C. Hecquet, J.-C. Jeanny, G. Soubrane, Y. Courtois, and F. Mascarelli Proliferation of CECs Requires Dual Signaling through Both MAPK/ERK and PI 3-K/Akt Pathways Invest. Ophthalmol. Vis. Sci., February 1, 2001; 42(2): 488 - 496. [Abstract] [Full Text]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
