September 2009
Volume 50, Issue 9
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Retinal Cell Biology  |   September 2009
Effects of Thrombin on RPE Cells Are Mediated by Transactivation of Growth Factor Receptors
Author Affiliations
  • Margrit Hollborn
    From the Department of Ophthalmology and Eye Hospital, and the
  • Carola Petto
    From the Department of Ophthalmology and Eye Hospital, and the
  • Anja Steffen
    From the Department of Ophthalmology and Eye Hospital, and the
    Interdisciplinary Center of Clinical Research (IZKF), University of Leipzig, Leipzig, Germany; and the
  • Susanne Trettner
    From the Department of Ophthalmology and Eye Hospital, and the
    Interdisciplinary Center of Clinical Research (IZKF), University of Leipzig, Leipzig, Germany; and the
  • Andrea Bendig
    From the Department of Ophthalmology and Eye Hospital, and the
    Interdisciplinary Center of Clinical Research (IZKF), University of Leipzig, Leipzig, Germany; and the
  • Peter Wiedemann
    From the Department of Ophthalmology and Eye Hospital, and the
  • Andreas Bringmann
    From the Department of Ophthalmology and Eye Hospital, and the
  • Leon Kohen
    From the Department of Ophthalmology and Eye Hospital, and the
    Helios Klinikum Aue, Aue, Germany.
Investigative Ophthalmology & Visual Science September 2009, Vol.50, 4452-4459. doi:10.1167/iovs.08-3194
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      Margrit Hollborn, Carola Petto, Anja Steffen, Susanne Trettner, Andrea Bendig, Peter Wiedemann, Andreas Bringmann, Leon Kohen; Effects of Thrombin on RPE Cells Are Mediated by Transactivation of Growth Factor Receptors. Invest. Ophthalmol. Vis. Sci. 2009;50(9):4452-4459. doi: 10.1167/iovs.08-3194.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To determine the expression of blood coagulation factors and thrombin receptors in retinal pigment epithelial (RPE) cells and whether the effects of thrombin on the chemotaxis and the secretion of VEGF are mediated by transactivation of growth factor receptors.

methods. Gene expression in acutely isolated and cultured human RPE cells was evaluated by RT-PCR. Alterations in gene expression and secretion of VEGF were determined by real-time RT-PCR and ELISA, respectively. Chemotaxis was examined with a Boyden chamber assay.

results. RPE cells expressed the mRNA of the protease-activated receptors PAR1 and -3 and of various coagulation factors (III, V, VII, VIII, and X). Thrombin stimulated the expression and secretion of VEGF-A from RPE cells, decreased the expression of VEGFD, and increased the gene expression of VEGFR-1 (FLT1). The effects on the secretion of VEGF-A and the increase in FLT1 expression were mediated by stimulation of the secretion of TGF-β1 and activation of the TGF-β activin receptor-like kinase. Thrombin stimulated the chemotaxis of RPE cells, and this effect was mediated by transactivation of the PDGF receptor tyrosine kinase.

conclusions. The expression of different coagulation factors suggests that RPE cells provide a procoagulant surface for the formation of thrombin from prothrombin via the extrinsic coagulation pathway. Thrombin stimulates the secretion of VEGF-A, the expression of FLT1, and the chemotaxis of RPE cells via transactivation of TGF-β and PDGF receptors, respectively.

A breakdown of the outer blood–retinal barrier constituted by the retinal pigment epithelium (RPE) is a characteristic event in the course of choroidal neovascularization and serous retinal detachment. 1 The vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors (Flt-1/VEGFR-1 and KDR/VEGFR-2) are key players in increasing the permeability of the blood–retinal barrier and in ocular neovascularization. 2 3 One major source of VEGF in the retina is the RPE. 4 RPE cells in situ secrete VEGF across their basolateral membranes, toward the choriocapillaris. 5 RPE-derived VEGF maintains the fenestrated permeable phenotype of the choriocapillaris endothelium and provides survival signals to endothelial cells. 6 7 Overexpression of VEGF in the RPE, in combination with a disruption of the RPE monolayer or Bruch’s membrane, has been shown to stimulate the development of choroidal neovascularization. 8 9 10 However, it has been shown that increased expression of VEGF in RPE cells alone is not sufficient to cause choroidal neovascularization, 11 suggesting that other factors (e.g., VEGF derived from other cells such as endothelial cells, a simultaneous decrease in the expression of antiangiogenic factors, or damage to Bruch’s membrane) are necessary for the angiogenic effect of VEGF. It has been suggested that, synergistically with VEGF, the actions of multiple proangiogenic factors, such as angiopoietin, insulin-like growth factor, platelet-derived growth factor (PDGF), transforming growth factor (TGF-β), fibroblast growth factor-2 (FGF-2), and placenta growth factor, are involved in the development of choroidal neovascularization. 10 11 12 13 14 15 16  
Another factor that acts synergistically with VEGF in promoting angiogenesis is the serine proteinase thrombin (coagulation factor IIa). 17 Thrombin is generated from prothrombin after contact to extravascular tissue factor (factor III), resulting in formation of fibrin from fibrinogen. 18 The development of choroidal neovascularization is often associated with the formation of a fibrin matrix. 10 19 Choroidal neovascular membranes have been described to be surrounded by a rim of fibrin, and it has been suggested that fibrin directs and possibly stimulates the growth and sprouting of newly formed vessels. 10 In choroidal neovascular membranes, fibrin deposition can also be found around the RPE. 19  
In addition to the formation of fibrin, thrombin promotes blood–retinal barrier breakdown and angiogenesis via a variety of cellular effects. It stimulates the VEGF-induced proliferation and migration of endothelial cells, which is associated with an upregulation of VEGF receptors in the cells. 20 Thrombin stimulates the secretion of growth factors and leukocyte chemotactic factors from RPE cells, and enhances the cell–cell contacts between RPE cells and monocytes. 21 22 23 Thrombin induces the formation of intercellular gaps in RPE monolayers, alters the distribution of cytoskeleton proteins, and promotes the expression of VEGF in RPE cells. 24 25 However, the intracellular signaling that mediates the stimulatory effect of thrombin on the expression of VEGF in RPE cells is incompletely understood. It is not known, for example, whether transactivation of receptors for growth factors, which are known to stimulate VEGF expression in RPE cells, 26 27 28 29 is involved in the effects of thrombin. Herein, we show that the thrombin-induced secretion (but not the expression) of VEGF-A is mediated by autocrine/paracrine TGF-β signaling. The transactivation of growth factor receptors is likely to be a more general phenomenon of thrombin action in RPE cells, since we found also that the thrombin-evoked chemotaxis is mediated by PDGF signaling. Furthermore, we determined whether RPE cells express blood coagulation factors that are implicated in the generation of thrombin from prothrombin and whether thrombin alters the expression of VEGF receptors in RPE cells. 
Materials and Methods
PD98059, LY294002, SP600125, Gö6976, ALLM, ALLN, and nimodipine were obtained from Calbiochem (Bad Soden, Germany); SB203580 from Tocris (Ellisville, MO); and recombinant TGF-β1, TGF-β2, and VEGF-A165 from R&D Systems (Wiesbaden, Germany). α-Thrombin, hirudin, staurosporine, and all other substances used were from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. At the concentrations used, the test substances had no effects on cell viability as determined by trypan blue staining (data not shown). The following polyclonal antibodies were used: a neutralizing panspecific rabbit anti-TGF-β directed against human TGF-β1, porcine TGF-β1 and -β2, and amphibian TGF-β5 (R&D Systems); a neutralizing goat anti-human heparin-binding epidermal growth factor-like (HB-EGF) antibody (R&D Systems); a neutralizing goat anti-human PDGF (R&D Systems); rabbit anti-human extracellular signal-regulated kinase-1 and -2 (ERK1/2, p44/p42; 1:1000; New England Biolabs, Frankfurt am Main, Germany); a rabbit anti-phosphorylated ERK1/2 (1:1000; New England Biolabs); a rabbit anti-human p38 mitogen-activated protein kinase (p38 MAPK, 1:1000; New England Biolabs); a rabbit anti-human phosphorylated p38 MAPK (1:750; New England Biolabs); and an anti-rabbit IgG conjugated with alkaline phosphatase (1:2000; Chemicon, Hofheim, Germany). 
Cell Culture
The use of human material was approved by the Ethics Committee of the University of Leipzig and was performed according to the Declaration of Helsinki. Human RPE cells were obtained from several donors within 48 hours of death, and were prepared and cultured as described in the Supplement
Real-Time PCR
The preparation of the total RNA from freshly isolated human RPE cells or from cultured cells, and the RT-PCR and real-time PCR analysis, were conducted by using the standard methods described in the Supplement. PCR was performed with primer pairs described in the Supplement. The mRNA expression was normalized to the levels of GAPDH mRNA. 
Enzyme-Linked Immunosorbent Assay
The cells were cultured at 3 × 103 cells per well in 96-well plates (100 μL culture medium per well). At a confluence of ∼80%, the cells were cultured in serum-free medium for 16 hours. Subsequently, the culture medium was changed, and the cells were stimulated with test substances, in the absence and presence of blocking substances. The supernatants were collected after 6 hours, and the level of VEGF-A165 or TGF-β1 in the culture medium (200 μL) was determined by ELISA (R&D Systems). 
Western Blot Analysis
The cells were seeded at 5 × 104 cells per well in six-well plates in 1.5 mL complete medium and were allowed to growth up to a confluence of ∼80%. After growth arrest for 16 hours, the cells were pretreated with blocking substances for 30 minutes and thereafter with test substances for 45 minutes. Then, the medium was removed, the cells were washed twice with prechilled phosphate-buffered saline (pH 7.4; Invitrogen, Paisley, UK), and the monolayer was scraped into 150 μL lysis buffer (Mammalian Cell Lysis-1 Kit; Sigma-Aldrich). The total cell lysates were centrifuged at 10,000g for 10 minutes, and the supernatants were analyzed in immunoblots. Equal amounts of protein (30 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis. The immunoblots were probed with primary and secondary antibodies, and immunoreactive bands were visualized with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. 
Chemotaxis
Chemotaxis was determined with a modified Boyden chamber assay as described in the Supplement. 
Statistics
Migration rates and the level of growth factors in the culture medium are expressed as a percentage of untreated control (100%). For each test, at least three independent experiments were performed in triplicate. Data were expressed as the mean ± SEM; statistical significance (Mann-Whitney U test and Kruskal-Wallis test followed by the Dunn comparison of multiple groups) was accepted at P < 0.05. 
Results
Expression of Coagulation Factors
RT-PCR analysis was performed to determine whether human RPE cells express mRNA for blood coagulation factors. The gene expression was analyzed in cultured cells and in cells that were freshly isolated from eyes obtained postmortem from four donors without apparent eye diseases. As shown in Figure 1A , freshly isolated cells expressed mRNA for the coagulation factors V, VII, VIII, and X. A similar expression pattern was found in cultured cells (Fig. 1B) . Both freshly isolated and cultured cells expressed the mRNA for the tissue factor (Figs. 1A 1B) . Under the conditions used, mRNA for prothrombin was expressed in five of the seven cell lines investigated, but was absent in cells freshly isolated from four different donors (not shown). mRNAs for fibrinogen-A and -B were absent in freshly isolated and cultured cells (not shown). 
Expression of Thrombin Receptors
We further investigated whether RPE cells express the mRNA of the thrombin receptors protease-activated receptor (PAR)-1, -3, and -4 and the receptor for trypsin and factor Xa, PAR2. We found that freshly isolated and cultured RPE cells expressed the mRNAs for PAR1 and -3, whereas the mRNAs for PAR2 and -4 were largely absent (Figs. 1A 1B) . The results are in agreement with previous studies that described expression of PAR1 and -3 in cultured RPE cells. 30 The data indicate that RPE cells express mRNA for various factors that are involved in the generation of thrombin from prothrombin, as well as for thrombin receptors. 
Regulation of VEGF and VEGF Receptor Expression by Thrombin
Exogenous thrombin caused alterations in the gene expression levels of VEGF-A and -D and of the VEGFR-1 (FLT1). As shown in Figure 2A , thrombin evoked an increase in the gene expression of VEGF-A which was significant (P < 0.05) after 2 hours of culturing. In addition, thrombin caused a decrease in the gene expression of VEGF-D that was significant (P < 0.05) at 4 and 6 hours of culturing. No differences in the mRNA levels for the VEGF proteins between stimulated and control cultures were observed after 24 hours of thrombin exposure (not shown). Thrombin increased the gene expression of VEGFR-1 (FLT1) and did not affect the expression level of VEGFR-2 (KDR; Fig. 2B ). Thrombin did not alter the gene expression of neuropilins-1 and -2 (NRP1 and -2) at each time period investigated (not shown). In contrast to thrombin, stimulation of the cells with VEGF-A165 caused a time-dependent increase in the gene expression of both FLT1 and KDR (Fig. 2C)
Thrombin-Evoked Secretion of VEGF-A
Since thrombin increased the mRNA expression of VEGF-A (Fig. 2A) , we investigated whether it also stimulates the secretion of VEGF-A protein by RPE cells. VEGF-A was constitutively released by the cells, and thrombin evoked a dose-dependent increase in the secretion of VEGF-A (Fig. 3A) . Thrombin at 10 U/mL approximately doubled the VEGF-A content of the culture supernatants. Hypoxia is a main inducer of VEGF-A in the retina. 31 We found that chemical hypoxia induced by culturing the cells in the presence of CoCl2 (150 μM) increased the secretion of VEGF-A from RPE cells (Fig. 3B) . The stimulatory effects of hypoxia and thrombin on the secretion of VEGF-A were additive (Fig. 3B) , suggesting that (partially) different signal transduction pathways are involved in mediating both effects. 
The thrombin-evoked secretion of VEGF-A was completely blocked by the inhibitor of thrombin enzymatic activity, hirudin (Fig. 3C) . The blocker of ERK1/2 activation, PD98059, as well as the inhibitor of p38 MAPK activation, SB203580, fully blocked the thrombin-evoked secretion of VEGF-A (Fig. 3C) . SB203580 decreased also the secretion of VEGF-A under control conditions, suggesting that the constitutive release of VEGF-A is mediated in part by activation of p38 MAPK. In addition, the effect of thrombin on the secretion of VEGF-A was blocked by an inhibitor of the c-Jun N-terminal kinase (JNK), SP600125 (Fig. 3D) . In contrast, an inhibitor of the phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, LY294002, was without effect (Fig. 3D) . Similarly, inhibitors of calpains I and II, of calcium-dependent isoforms of protein kinase C, and a broad spectrum inhibitor of protein kinases did not reduce the thrombin-evoked secretion of VEGF-A (Fig. 3D) . Moreover, incubation of the cells with nimodipine (10 μM), a blocker of L-type voltage-dependent calcium channels, did not alter the effect of thrombin (not shown). The data suggest that the stimulatory effect of thrombin on the secretion of VEGF-A by RPE cells is mediated by activation of the ERK1/2, p38, and JNK signal transduction pathways. Using Western blot analysis, we found that thrombin enhanced dose dependently the degree of phosphorylation of the ERK1/2 and p38 proteins in RPE cells (Fig. 3E)
Involvement of TGF-β in the Effects of Thrombin
It has been shown that the secretion of VEGF from vascular smooth muscle cells in response to thrombin is mediated in part by a thrombin-induced release of growth factors—for example, TGF-β. 32 Since TGF-β is a known inducer of VEGF-A in RPE cells, 26 we tested whether the effects of thrombin are mediated by stimulation of TGF-β release from RPE cells. As shown in Figure 4A , thrombin evoked an increase in the gene expression of various growth factors such as PDGF, FGF-2, and HB-EGF, whereas the mRNA expression of TGFB1 and hepatocyte growth factor remained unaltered. However, thrombin induced secretion of TGF-β1 from RPE cells, as indicated by the elevation of the TGF-β1 protein content of the culture medium (Fig. 4B) . The thrombin-evoked increase in the VEGF-A content of the cultured medium was abrogated in the presence of a neutralizing anti-TGF-β antibody (Fig. 4C)and of a selective blocker of the TGF-β activin receptor-like kinase SB431542, (Fig. 4D) , respectively. On the other hand, the anti-TGF-β antibody had no effect on the thrombin (10 U/mL)-induced elevation in the mRNA expression of VEGF-A (not shown). The stimulatory effect of thrombin on the mRNA expression of FLT1 was also abrogated in the presence of a neutralizing anti-TGF-β antibody (Fig. 2B) . As the control, we tested the anti-inflammatory corticosteroid triamcinolone acetonide which is known to inhibit the secretion of VEGF-A by RPE cells. 33 34 Triamcinolone decreased the VEGF-A content of the culture medium significantly in the absence and presence of thrombin (Fig. 4C)
Since thrombin stimulated the expression of multiple growth factors (Fig. 4A) , we tested various other neutralizing antibodies and receptor activation-blocking substances. Inhibition of HB-EGF and PDGF signaling (either by neutralizing anti-HB-EGF and anti-PDGF antibodies or by selective blockers of the EGF and PDGF receptor tyrosine kinases) had no effects on the thrombin-induced stimulation of VEGF-A secretion from RPE cells (Fig. 4D) . Similarly, the selective inhibitor of KDR SU1498 (10 μM) did not prevent the effect of thrombin (10 U/mL) on the secretion of VEGF-A (not shown). 1,10-Phenanthroline (10 μM), a broad-spectrum metalloproteinase inhibitor, was also without effect (not shown). 
Thrombin-Evoked Chemotaxis
Thrombin stimulated dose dependently the chemotaxis of RPE cells, with a half-maximum effect at ∼3 U/mL (Fig. 5A) . The motogenic effect of thrombin was prevented by selective inhibitors of the PDGF receptor tyrosine kinase and p38 MAPK activation (Fig. 5B) . Thrombin-induced chemotaxis was not inhibited by blocking the activation of ERK1/2 or JNK (Fig. 5B) , by a neutralizing anti-TGF-β antibody (20 μg/mL; not shown), and by the selective blocker of KDR SU1498 (10 μM; not shown). The data suggest that thrombin-induced chemotaxis is mediated by transactivation of the PDGF receptor tyrosine kinase and the p38 MAPK signal transduction pathway. Triamcinolone acetonide fully inhibited thrombin-induced chemotaxis (Fig. 5B)
Discussion
We found that RPE cells express mRNA for various factors which are implicated in the generation of thrombin from prothrombin, suggesting that the cells provide a procoagulant surface. This assumption is supported by the following observations: (1) RPE cells express the mRNAs for factors V and X (Fig. 1A) . Factor Xa and its cofactor Va form the prothrombinase complex that generates thrombin from prothrombin. 35 36 (2) RPE cells express factors that are implicated in the activation of factor X. Factor X can be activated by the intrinsic and extrinsic pathways of blood coagulation. The factor X-activating complex, that plays a key role in the intrinsic pathway, consists of the enzyme factor IXa, the substrate factor X, and the cofactor factor VIIIa. 37 However, we found that RPE cells do not express mRNA for factor IX (Fig. 1) , which may exclude the possibility that this pathway plays a major role in the activation of factor X by RPE cells. On the other hand, RPE cells express mRNAs for factor VII and tissue factor (Fig. 1) . Factor X can be directly activated by factor VIIa which forms a complex with its receptor, tissue factor (which is the primary cellular initiator of the coagulation cascade after disruption of vascular integrity), and factor X. 18 38 The data suggest that RPE cells are capable of generating thrombin from prothrombin via the extrinsic coagulation pathway. The absence of mRNA for prothrombin in cells freshly isolated from donor eyes suggests that RPE cells in situ may produce thrombin predominantly from serum-derived prothrombin which may enter the retina from the choroidal circulation. Whether RPE cells in situ are capable of producing prothrombin in pathologic conditions remains to be determined. 
In addition to the generation of fibrin, thrombin has various other effects on RPE cells. RPE cells express the mRNA for two thrombin receptors, PAR1 and -3 (Fig. 1) . Since PAR3 is not activated by thrombin but binds this enzyme and acts as cofactor, which is, for example, necessary for the activation of PAR4 by thrombin, 39 we assume that the effects of thrombin in RPE cells are predominantly mediated by PAR1, as previously suggested for the thrombin-induced VEGF-A expression. 25 In the present study, we describe that thrombin induces mRNA expression and secretion of VEGF-A, increases the expression of FLT1, and stimulates chemotaxis of RPE cells. The thrombin-induced release of VEGF-A is mediated by activation of ERK1/2, p38, and JNK signal transduction pathways; activation of p38 contributes to the stimulation of chemotaxis. We did not find evidence of an involvement of PI3K-Akt or protein kinase C activation in mediating the stimulatory effect of thrombin on the secretion of VEGF-A. A calcium-independent, but ERK1/2-dependent release of VEGF-A on stimulation of PAR1 receptors has been described in fibroblasts. 40 The inhibitors of ERK1/2 and JNK activation, PD98059 and SP600125, slightly stimulated the chemotaxis of RPE cells in control conditions (Fig. 5B) . The mechanism of this effect is unclear, but may be explained (at least in part) by the antagonism between cellular proliferation and migration which is mediated by an inhibitory cross-talk between p38 and ERK1/2. This antagonism was described in various cell systems (e.g., RPE cells). 28  
We found that distinct effects of thrombin in RPE cells are dependent on transactivation of growth factor receptors. The thrombin-induced secretion of VEGF-A and the expression of FLT1 are dependent on autocrine/paracrine TGF-β signaling, whereas thrombin-evoked chemotaxis is dependent on PDGF signaling. The thrombin-induced secretion of VEGF-A is mediated by stimulation of the secretion (but not expression) of TGF-β from the cells and subsequent activation of the TGF-β activin receptor-like kinase. The involvement of autocrine/paracrine TGF-β signaling was approved by two different methods: by using a neutralizing anti-TGF-β antibody (Fig. 4C)and by using a pharmacologic inhibitor of the TGF-β activin receptor-like kinase (Fig. 4D) . Although it cannot be completely excluded, it seems unlikely that unspecific effects of the pharmacologic blocker causes the inhibition of the thrombin effect. Apparently, the effects of thrombin on the expression and secretion of VEGF-A are mediated by different signal transduction pathways, with only the latter depending on TGF-β signaling. 
Thrombin evoked an enhanced mRNA expression of various growth factors, such as PDGF, FGF-2, and HB-EGF, and a secretion of TGF-β1 from RPE cells. The strongest elevation in gene expression was found for HB-EGF (Fig. 4A) . Although HB-EGF is known to induce secretion of VEGF-A from RPE cells, 27 29 we did not find an involvement of this factor in the thrombin-induced VEGF-A secretion. The lack of HB-EGF action can be explained by the absence of metalloproteinase activation after treatment with thrombin. HB-EGF must be shed from its membrane-bound precursor (pro-HB-EGF) by matrix metalloproteinases before activation of the EGF receptor. 41 Although thrombin may not activate matrix metalloproteinases, the thrombin-induced increase in HB-EGF expression may contribute to the increase in tissue VEGF-A in in situ conditions when the RPE cell’s matrix metalloproteinases are activated by other factors. 
Thrombin exerts multiple effects on RPE cells that could be involved in the pathogenesis of subretinal edema and neovascularization. 21 22 23 24 25 Among others, thrombin stimulates the gene expression or secretion of the angiogenic factors VEGF-A, FGF-2, TGF-β, and PDGF by RPE cells. The thrombin-evoked upregulation of FLT1 may be involved in promotion or inhibition of choroidal neovascularization. 25 42 43 We found that triamcinolone acetonide prevents the thrombin-evoked secretion of VEGF-A and migration of RPE cells. It is known that PARs transmit proinflammatory signals including the expression of inflammatory and growth factors, 44 and that anticoagulant therapy attenuates inflammatory responses and fibrin formation in the retina. 45 46 47 It is suggested that the therapeutic action of triamcinolone in retinal edema and neovascularization is mediated in part by the inhibitory effect on the action of thrombin. Thrombin is used as a therapeutic agent, for example, for the closure of macular holes 48 ; this effect of thrombin may be mediated by the autocrine/paracrine mitogenic 21 22 and motogenic action of PDGF (Fig. 5A)
In summary, in our study RPE cells provided a procoagulant surface for the formation of thrombin from prothrombin. Thrombin induces the expression or release of various proangiogenic factors from RPE cells, increases FLT1, and stimulates chemotaxis. The stimulatory effect of thrombin on the secretion of the major angiogenic factor VEGF-A is mediated by autocrine/paracrine TGF-β signaling, whereas thrombin-evoked chemotaxis is mediated by PDGF signaling. Upregulation of angiogenic factors may represent an important mechanism by which the coagulation cascade contributes to subretinal edema and neovascularization. 
 
Figure 1.
 
Gene expression of blood coagulation factors and thrombin receptors in human RPE cells, as determined by RT-PCR. (A) Freshly isolated cells from two different donors (1 and 2). (B) Cultured cells of passages 5 (donor 1) and 4 (donor 2). In the negative control (−) experiments double-distilled water instead of cDNA was added as a template.
Figure 1.
 
Gene expression of blood coagulation factors and thrombin receptors in human RPE cells, as determined by RT-PCR. (A) Freshly isolated cells from two different donors (1 and 2). (B) Cultured cells of passages 5 (donor 1) and 4 (donor 2). In the negative control (−) experiments double-distilled water instead of cDNA was added as a template.
Figure 2.
 
Thrombin alters the gene expression of VEGF and FLT1 in human RPE cells. (A) Thrombin increased transiently the expression of VEGFA and decreased the expression of VEGFD. The mRNA levels of various members of the VEGF family of growth factors were determined by real-time RT-PCR after 2-, 4-, and 6-hour stimulation of the cells with thrombin (10 U/mL). (B) Thrombin (10 U/mL) increased the expression of VEGF-R1 (FLT1) and had no effect on the expression of VEGF-R2 (KDR). The stimulatory effect of thrombin on the expression of FLT1 was prevented in the presence of a neutralizing antibody against TGF-β (aTGF-β; 20 μg/mL). Administration of the antibody alone had no effect on the expression of FLT1 and KDR (not shown). (C) VEGF-A165 (10 ng/mL) evoked increases in the expression of FLT1 and KDR. The diagrams display the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus unstimulated control: *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 2.
 
Thrombin alters the gene expression of VEGF and FLT1 in human RPE cells. (A) Thrombin increased transiently the expression of VEGFA and decreased the expression of VEGFD. The mRNA levels of various members of the VEGF family of growth factors were determined by real-time RT-PCR after 2-, 4-, and 6-hour stimulation of the cells with thrombin (10 U/mL). (B) Thrombin (10 U/mL) increased the expression of VEGF-R1 (FLT1) and had no effect on the expression of VEGF-R2 (KDR). The stimulatory effect of thrombin on the expression of FLT1 was prevented in the presence of a neutralizing antibody against TGF-β (aTGF-β; 20 μg/mL). Administration of the antibody alone had no effect on the expression of FLT1 and KDR (not shown). (C) VEGF-A165 (10 ng/mL) evoked increases in the expression of FLT1 and KDR. The diagrams display the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus unstimulated control: *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 3.
 
Thrombin stimulates the secretion of VEGF-A by human RPE cells. The cells were cultured for 6 hours in the presence of thrombin, and the concentration of VEGF-A165 in the culture supernatants was measured by ELISA. (A) Dose dependence of the effect of thrombin on the amount of VEGF-A protein in the cultured medium. Concentrations of thrombin (in U/mL) are given in the histograms. (B) Effects of chemical hypoxia, evoked by CoCl2 (150 μM) and thrombin (10 U/mL) on the VEGF-A content of the culture medium. (C) Effect of various blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: the thrombin inhibitor hirudin (20 U/mL), the MEK inhibitor PD98059 (20 μM), and the inhibitor of p38 activation SB203580 (10 μM). (D) The stimulatory effect of thrombin (10 U/mL) on the secretion of VEGF-A was blocked by the JNK inhibitor SP600125 (10 μM). The effect of thrombin remained unaltered in the presence of the inhibitor of PI3K LY294002 (5 μM), the calpain I and II inhibitors ALLM (500 nM) and ALLN (500 nM), the inhibitor of calcium-dependent isoforms of protein kinase C Gö6976 (1 μM), and the broad-spectrum inhibitor of protein kinases, staurosporine (10 nM). (E) Thrombin enhanced dose dependently the cellular level of phosphorylated ERK1/2 and p38 proteins, as revealed by Western blot analysis. The total amount of both proteins remained unchanged. The cultures were stimulated with thrombin for 10 minutes. In (AD), the cells were incubated with test substances for 6 hours; blocking substances were preincubated for 1 hour. Data are expressed as the percentage of untreated control (100%) which was between 100 and 250 pg/mL. Results are expressed as the mean ± SEM of three to seven independent experiments on cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ○P < 0.05.
Figure 3.
 
Thrombin stimulates the secretion of VEGF-A by human RPE cells. The cells were cultured for 6 hours in the presence of thrombin, and the concentration of VEGF-A165 in the culture supernatants was measured by ELISA. (A) Dose dependence of the effect of thrombin on the amount of VEGF-A protein in the cultured medium. Concentrations of thrombin (in U/mL) are given in the histograms. (B) Effects of chemical hypoxia, evoked by CoCl2 (150 μM) and thrombin (10 U/mL) on the VEGF-A content of the culture medium. (C) Effect of various blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: the thrombin inhibitor hirudin (20 U/mL), the MEK inhibitor PD98059 (20 μM), and the inhibitor of p38 activation SB203580 (10 μM). (D) The stimulatory effect of thrombin (10 U/mL) on the secretion of VEGF-A was blocked by the JNK inhibitor SP600125 (10 μM). The effect of thrombin remained unaltered in the presence of the inhibitor of PI3K LY294002 (5 μM), the calpain I and II inhibitors ALLM (500 nM) and ALLN (500 nM), the inhibitor of calcium-dependent isoforms of protein kinase C Gö6976 (1 μM), and the broad-spectrum inhibitor of protein kinases, staurosporine (10 nM). (E) Thrombin enhanced dose dependently the cellular level of phosphorylated ERK1/2 and p38 proteins, as revealed by Western blot analysis. The total amount of both proteins remained unchanged. The cultures were stimulated with thrombin for 10 minutes. In (AD), the cells were incubated with test substances for 6 hours; blocking substances were preincubated for 1 hour. Data are expressed as the percentage of untreated control (100%) which was between 100 and 250 pg/mL. Results are expressed as the mean ± SEM of three to seven independent experiments on cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ○P < 0.05.
Figure 4.
 
The stimulatory effect of thrombin on the secretion of VEGF-A from RPE cells is mediated by autocrine TGF-β signaling. (A) Thrombin increased the expression of VEGFA, PDGF, FGF2, and HBEGF, but not of TGFB1 and HGF. The mRNA levels of growth factors were determined by real-time RT-PCR after 2- and 24-hour stimulations of the cells with thrombin (10 U/mL). (B) Thrombin increased the secretion of TGF-β1 from RPE cells. The histograms display the relative TGF-β1 protein content in the media from cultures that were stimulated for 6 or 24 hours with thrombin (10 U/mL), as measured by ELISA. (C) Effects of TGF-β1 and -β2 (10 ng/mL each), a neutralizing anti-TGF-β antibody (aTGF-β; 20 μg/mL), and triamcinolone acetonide (Triam; 50 μM), on the secretion of VEGF-A. (D) Effect of blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: a neutralizing anti-HB-EGF antibody (aHB-EGF; 10 μg/mL); a neutralizing anti-PDGF antibody (aPDGF; 10 μg/mL); the selective inhibitor of the TGF-β activin receptor-like kinase, SB431542 (10 μM); the selective blocker of the EGF receptor tyrosine kinase, tyrphostin AG1478 (600 nM); and the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM). (C, D) The agents were tested in the absence and presence of thrombin (10 U/mL) for 6 hours, and the level of VEGF-A in the cultured media was determined by ELISA. Results are expressed as the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus control: *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 4.
 
The stimulatory effect of thrombin on the secretion of VEGF-A from RPE cells is mediated by autocrine TGF-β signaling. (A) Thrombin increased the expression of VEGFA, PDGF, FGF2, and HBEGF, but not of TGFB1 and HGF. The mRNA levels of growth factors were determined by real-time RT-PCR after 2- and 24-hour stimulations of the cells with thrombin (10 U/mL). (B) Thrombin increased the secretion of TGF-β1 from RPE cells. The histograms display the relative TGF-β1 protein content in the media from cultures that were stimulated for 6 or 24 hours with thrombin (10 U/mL), as measured by ELISA. (C) Effects of TGF-β1 and -β2 (10 ng/mL each), a neutralizing anti-TGF-β antibody (aTGF-β; 20 μg/mL), and triamcinolone acetonide (Triam; 50 μM), on the secretion of VEGF-A. (D) Effect of blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: a neutralizing anti-HB-EGF antibody (aHB-EGF; 10 μg/mL); a neutralizing anti-PDGF antibody (aPDGF; 10 μg/mL); the selective inhibitor of the TGF-β activin receptor-like kinase, SB431542 (10 μM); the selective blocker of the EGF receptor tyrosine kinase, tyrphostin AG1478 (600 nM); and the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM). (C, D) The agents were tested in the absence and presence of thrombin (10 U/mL) for 6 hours, and the level of VEGF-A in the cultured media was determined by ELISA. Results are expressed as the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus control: *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 5.
 
Thrombin evoked chemotaxis of RPE cells. (A) Concentration-dependent migration of RPE cells evoked by thrombin. Data are the mean ± SEM obtained in five independent experiments. (B) The thrombin (10 U/mL)-evoked chemotaxis was prevented in the presence of the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM); the inhibitor of p38 MAPK activation, SB203580 (10 μM); and triamcinolone acetonide (Triam; 10 μM). The MEK inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) did not inhibit thrombin-evoked chemotaxis. Results are expressed as the mean ± SEM of five and eight independent experiments using cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05.
Figure 5.
 
Thrombin evoked chemotaxis of RPE cells. (A) Concentration-dependent migration of RPE cells evoked by thrombin. Data are the mean ± SEM obtained in five independent experiments. (B) The thrombin (10 U/mL)-evoked chemotaxis was prevented in the presence of the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM); the inhibitor of p38 MAPK activation, SB203580 (10 μM); and triamcinolone acetonide (Triam; 10 μM). The MEK inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) did not inhibit thrombin-evoked chemotaxis. Results are expressed as the mean ± SEM of five and eight independent experiments using cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05.
Supplementary Materials
Supplement - (PDF) 
The authors thank Ute Weinbrecht and Franziska Rickers for excellent technical support. 
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Figure 1.
 
Gene expression of blood coagulation factors and thrombin receptors in human RPE cells, as determined by RT-PCR. (A) Freshly isolated cells from two different donors (1 and 2). (B) Cultured cells of passages 5 (donor 1) and 4 (donor 2). In the negative control (−) experiments double-distilled water instead of cDNA was added as a template.
Figure 1.
 
Gene expression of blood coagulation factors and thrombin receptors in human RPE cells, as determined by RT-PCR. (A) Freshly isolated cells from two different donors (1 and 2). (B) Cultured cells of passages 5 (donor 1) and 4 (donor 2). In the negative control (−) experiments double-distilled water instead of cDNA was added as a template.
Figure 2.
 
Thrombin alters the gene expression of VEGF and FLT1 in human RPE cells. (A) Thrombin increased transiently the expression of VEGFA and decreased the expression of VEGFD. The mRNA levels of various members of the VEGF family of growth factors were determined by real-time RT-PCR after 2-, 4-, and 6-hour stimulation of the cells with thrombin (10 U/mL). (B) Thrombin (10 U/mL) increased the expression of VEGF-R1 (FLT1) and had no effect on the expression of VEGF-R2 (KDR). The stimulatory effect of thrombin on the expression of FLT1 was prevented in the presence of a neutralizing antibody against TGF-β (aTGF-β; 20 μg/mL). Administration of the antibody alone had no effect on the expression of FLT1 and KDR (not shown). (C) VEGF-A165 (10 ng/mL) evoked increases in the expression of FLT1 and KDR. The diagrams display the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus unstimulated control: *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 2.
 
Thrombin alters the gene expression of VEGF and FLT1 in human RPE cells. (A) Thrombin increased transiently the expression of VEGFA and decreased the expression of VEGFD. The mRNA levels of various members of the VEGF family of growth factors were determined by real-time RT-PCR after 2-, 4-, and 6-hour stimulation of the cells with thrombin (10 U/mL). (B) Thrombin (10 U/mL) increased the expression of VEGF-R1 (FLT1) and had no effect on the expression of VEGF-R2 (KDR). The stimulatory effect of thrombin on the expression of FLT1 was prevented in the presence of a neutralizing antibody against TGF-β (aTGF-β; 20 μg/mL). Administration of the antibody alone had no effect on the expression of FLT1 and KDR (not shown). (C) VEGF-A165 (10 ng/mL) evoked increases in the expression of FLT1 and KDR. The diagrams display the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus unstimulated control: *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 3.
 
Thrombin stimulates the secretion of VEGF-A by human RPE cells. The cells were cultured for 6 hours in the presence of thrombin, and the concentration of VEGF-A165 in the culture supernatants was measured by ELISA. (A) Dose dependence of the effect of thrombin on the amount of VEGF-A protein in the cultured medium. Concentrations of thrombin (in U/mL) are given in the histograms. (B) Effects of chemical hypoxia, evoked by CoCl2 (150 μM) and thrombin (10 U/mL) on the VEGF-A content of the culture medium. (C) Effect of various blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: the thrombin inhibitor hirudin (20 U/mL), the MEK inhibitor PD98059 (20 μM), and the inhibitor of p38 activation SB203580 (10 μM). (D) The stimulatory effect of thrombin (10 U/mL) on the secretion of VEGF-A was blocked by the JNK inhibitor SP600125 (10 μM). The effect of thrombin remained unaltered in the presence of the inhibitor of PI3K LY294002 (5 μM), the calpain I and II inhibitors ALLM (500 nM) and ALLN (500 nM), the inhibitor of calcium-dependent isoforms of protein kinase C Gö6976 (1 μM), and the broad-spectrum inhibitor of protein kinases, staurosporine (10 nM). (E) Thrombin enhanced dose dependently the cellular level of phosphorylated ERK1/2 and p38 proteins, as revealed by Western blot analysis. The total amount of both proteins remained unchanged. The cultures were stimulated with thrombin for 10 minutes. In (AD), the cells were incubated with test substances for 6 hours; blocking substances were preincubated for 1 hour. Data are expressed as the percentage of untreated control (100%) which was between 100 and 250 pg/mL. Results are expressed as the mean ± SEM of three to seven independent experiments on cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ○P < 0.05.
Figure 3.
 
Thrombin stimulates the secretion of VEGF-A by human RPE cells. The cells were cultured for 6 hours in the presence of thrombin, and the concentration of VEGF-A165 in the culture supernatants was measured by ELISA. (A) Dose dependence of the effect of thrombin on the amount of VEGF-A protein in the cultured medium. Concentrations of thrombin (in U/mL) are given in the histograms. (B) Effects of chemical hypoxia, evoked by CoCl2 (150 μM) and thrombin (10 U/mL) on the VEGF-A content of the culture medium. (C) Effect of various blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: the thrombin inhibitor hirudin (20 U/mL), the MEK inhibitor PD98059 (20 μM), and the inhibitor of p38 activation SB203580 (10 μM). (D) The stimulatory effect of thrombin (10 U/mL) on the secretion of VEGF-A was blocked by the JNK inhibitor SP600125 (10 μM). The effect of thrombin remained unaltered in the presence of the inhibitor of PI3K LY294002 (5 μM), the calpain I and II inhibitors ALLM (500 nM) and ALLN (500 nM), the inhibitor of calcium-dependent isoforms of protein kinase C Gö6976 (1 μM), and the broad-spectrum inhibitor of protein kinases, staurosporine (10 nM). (E) Thrombin enhanced dose dependently the cellular level of phosphorylated ERK1/2 and p38 proteins, as revealed by Western blot analysis. The total amount of both proteins remained unchanged. The cultures were stimulated with thrombin for 10 minutes. In (AD), the cells were incubated with test substances for 6 hours; blocking substances were preincubated for 1 hour. Data are expressed as the percentage of untreated control (100%) which was between 100 and 250 pg/mL. Results are expressed as the mean ± SEM of three to seven independent experiments on cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01. Significant blocking effects: •P < 0.05; ○P < 0.05.
Figure 4.
 
The stimulatory effect of thrombin on the secretion of VEGF-A from RPE cells is mediated by autocrine TGF-β signaling. (A) Thrombin increased the expression of VEGFA, PDGF, FGF2, and HBEGF, but not of TGFB1 and HGF. The mRNA levels of growth factors were determined by real-time RT-PCR after 2- and 24-hour stimulations of the cells with thrombin (10 U/mL). (B) Thrombin increased the secretion of TGF-β1 from RPE cells. The histograms display the relative TGF-β1 protein content in the media from cultures that were stimulated for 6 or 24 hours with thrombin (10 U/mL), as measured by ELISA. (C) Effects of TGF-β1 and -β2 (10 ng/mL each), a neutralizing anti-TGF-β antibody (aTGF-β; 20 μg/mL), and triamcinolone acetonide (Triam; 50 μM), on the secretion of VEGF-A. (D) Effect of blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: a neutralizing anti-HB-EGF antibody (aHB-EGF; 10 μg/mL); a neutralizing anti-PDGF antibody (aPDGF; 10 μg/mL); the selective inhibitor of the TGF-β activin receptor-like kinase, SB431542 (10 μM); the selective blocker of the EGF receptor tyrosine kinase, tyrphostin AG1478 (600 nM); and the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM). (C, D) The agents were tested in the absence and presence of thrombin (10 U/mL) for 6 hours, and the level of VEGF-A in the cultured media was determined by ELISA. Results are expressed as the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus control: *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 4.
 
The stimulatory effect of thrombin on the secretion of VEGF-A from RPE cells is mediated by autocrine TGF-β signaling. (A) Thrombin increased the expression of VEGFA, PDGF, FGF2, and HBEGF, but not of TGFB1 and HGF. The mRNA levels of growth factors were determined by real-time RT-PCR after 2- and 24-hour stimulations of the cells with thrombin (10 U/mL). (B) Thrombin increased the secretion of TGF-β1 from RPE cells. The histograms display the relative TGF-β1 protein content in the media from cultures that were stimulated for 6 or 24 hours with thrombin (10 U/mL), as measured by ELISA. (C) Effects of TGF-β1 and -β2 (10 ng/mL each), a neutralizing anti-TGF-β antibody (aTGF-β; 20 μg/mL), and triamcinolone acetonide (Triam; 50 μM), on the secretion of VEGF-A. (D) Effect of blocking substances on the thrombin (10 U/mL)-evoked secretion of VEGF-A. The following substances were tested: a neutralizing anti-HB-EGF antibody (aHB-EGF; 10 μg/mL); a neutralizing anti-PDGF antibody (aPDGF; 10 μg/mL); the selective inhibitor of the TGF-β activin receptor-like kinase, SB431542 (10 μM); the selective blocker of the EGF receptor tyrosine kinase, tyrphostin AG1478 (600 nM); and the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM). (C, D) The agents were tested in the absence and presence of thrombin (10 U/mL) for 6 hours, and the level of VEGF-A in the cultured media was determined by ELISA. Results are expressed as the mean ± SEM of three to five independent experiments using cells from different donors. Significant differences versus control: *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05; ••P < 0.01.
Figure 5.
 
Thrombin evoked chemotaxis of RPE cells. (A) Concentration-dependent migration of RPE cells evoked by thrombin. Data are the mean ± SEM obtained in five independent experiments. (B) The thrombin (10 U/mL)-evoked chemotaxis was prevented in the presence of the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM); the inhibitor of p38 MAPK activation, SB203580 (10 μM); and triamcinolone acetonide (Triam; 10 μM). The MEK inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) did not inhibit thrombin-evoked chemotaxis. Results are expressed as the mean ± SEM of five and eight independent experiments using cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05.
Figure 5.
 
Thrombin evoked chemotaxis of RPE cells. (A) Concentration-dependent migration of RPE cells evoked by thrombin. Data are the mean ± SEM obtained in five independent experiments. (B) The thrombin (10 U/mL)-evoked chemotaxis was prevented in the presence of the selective inhibitor of the PDGF receptor tyrosine kinase, tyrphostin AG1296 (10 μM); the inhibitor of p38 MAPK activation, SB203580 (10 μM); and triamcinolone acetonide (Triam; 10 μM). The MEK inhibitor PD98059 (20 μM), and the JNK inhibitor SP600125 (10 μM) did not inhibit thrombin-evoked chemotaxis. Results are expressed as the mean ± SEM of five and eight independent experiments using cells from different donors. Significant differences versus control (100%): *P < 0.05; **P < 0.01; ***P < 0.001. Significant blocking effects: •P < 0.05.
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