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CARDIOVASCULAR
Laboratory of Cellular Tissue Engineering, School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania (J.-P.D., A.R., F.D.A., P.L., P.I.L.); and Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel (P.L.)
Received July 25, 2005; accepted August 23, 2005.
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Abstract |
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0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by 30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(|)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system.
). In response to a stimulus by an angiogenic growth factor, endothelial cells migrate into the interstitial space by first degrading the underlying basement membrane. Behind the front of migrating cells, other endothelial cells continuously proliferate to provide the necessary number of cells to generate the new vessel (neoangiogenesis) (Ausprunk et al., 1974).
Both migration and proliferation of endothelial cells (ECs) are pharmacological targets for drug discovery (Cai et al., 2000). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are known to induce migration and proliferation of endothelial cells in vitro and in vivo (Rousseau et al., 2000; Poole et al., 2001). These, like most other effects of VEGF and FGF, are mediated by activation of tyrosine kinase receptors Flk-1/KDR (VEGF) and FGF receptors (FGF), respectively, which leads to the phosphorylation of a variety of downstream targets, including cytoskeletal proteins, which in turn regulate EC migration (Kanda et al., 2004).
NGF is an evolutionary conserved polypeptide of the neurotrophin family that plays a crucial role in the life of the sympathetic and sensory nervous systems (Levi-Montalcini, 1987). The majority of research on NGF has been performed using NGF isolated from the male mouse submaxillary gland. More recently, human recombinant NGF (Rask, 1999) and snake venom NGFs (Hayashi et al., 1996; Katzir et al., 2003) have become important additional NGF agonist tools.
Several recent reports indicate that NGF exerts a variety of effects on peripheral tissues, including the vasculature, suggesting that NGF may be a novel angiogenic factor (Cantarella et al., 2002; Lazarovici et al., 2005). In studying the effects of NGFs on EC migration, we used two different aortic ECs isolated from humans (human aortic endothelial cells, HAECs) and rats (rat aortic endothelial cells, RAOECs). Both these cell lines had been previously characterized in our laboratory for adhesion molecules and expression of various adenylate cyclase isoforms (Manolopoulos et al., 1995; Kanda et al., 1998). For comparison, we also used rat adrenal medullary endothelial cells (RAMECs), which respond to thrombin stimulation by secretion of different extracellular matrix proteins (Papadimitriou et al., 1997).
To investigate the effects of NGF on EC migration, we modified and optimized a previously described omnidirectional migration (OM) assay (Cai et al., 2000). Using this assay, we demonstrated that NGF from various species induced migration of EC cells, albeit with various efficacies. The effects of viper-NGF (vNGF) were comparable with those induced by human recombinant VEGF (rhVEGF) but were less efficacious than the strong migratory effect elicited by human recombinant bFGF (rhbFGF). NGF-induced EC migration was selectively blocked by K252a (NGF receptor inhibitor) but not by SU-5416 (VEGF receptor inhibitor). These results strongly support the concept that NGF may represent a novel angiogenic factor, which among other angiogenic effects induces migration of cultured aortic endothelial cells.
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Materials and Methods |
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Growth Factors. Human recombinant epidermal growth factor and vascular endothelial growth factor (rhVEGF 165) were purchased from Sigma-Aldrich. Human recombinant basic fibroblast growth factor (rhbFGF) was kindly provided by Cytolab Co. (Rehovot, Israel). Human recombinant nerve growth factor (rhNGF) and mouse 
-nerve growth factor (2.5S-mNGF) were kindly supplied by Alomone Labs (Jerusalem, Israel). vNGF was purified as described previously (Hayashi et al., 1996; Katzir et al., 2003). Stock solutions of the different NGFs were routinely analyzed for activity in the PC12 bioassay (Katzir et al., 2003). Stock solutions of all growth factors (0.2–2.2 mg/ml) in PBS were aliquoted and stored at –20°C.
Drugs. The high-affinity NGF-receptor (trk) antagonist K252a was a gift from Kyowa Hakko Kogyo Co. (Tokyo, Japan). The selective VEGF receptor (Flk-1/KDR) antagonist SU-5416 was kindly provided by Dr. Aviv Gazit (Department of Organic Chemistry, The Hebrew University of Jerusalem, Israel). The drugs were dissolved in DMSO at concentrations of 1 mM (K252a) and 10 mM (SU-5416), aliquoted, and kept in the dark at –20°C.
Cell Culture. Several previously described EC lines were used in this study: RAMECs (Papadimitriou et al., 1997), RAOECs (Manolopoulos et al., 1995), and HAECs (Kanda et al., 1998). All cell lines were adapted to grow in a common growth medium (GM) composed of MCDB-131 and M-199 at a ratio of 1:1 supplemented with 7.5% (w/v) sodium bicarbonate, 10% FBS, 50 IU of penicillin, 50 µg/ml streptomycin, 25 µg/ml amphotericin B, 2 mM L-alanyl-L-glutamine, 2.3 µM hydrocortisone, 10 U/ml heparin, 10 ng/ml human recombinant epidermal growth factor, and 3 ng/ml rhbFGF at pH 7.4. The cells were grown in a humidified tissue culture incubator in 5% CO2 at 37°C. For the migration experiments, we used EC cultures between passages 10 and 24. In terms of their migratory capabilities, the cells did not display any significant differences between early and late passages.
OM Assay. For this work, we modified and optimized the OM assay recently described by Dixit et al. (2001). In brief, in the first stage (Fig. 1A, step 1), we marked the outside bottom surface of six-well tissue culture plates (BD Biosciences, San Jose, CA) denoting the outer circumference of a cloning ring (4 mm in diameter; Fisher Scientific Co., Pittsburgh, PA). The center of the ring was also marked to allow for precise positioning of the ring inside the well before cell application and later on for accurate photography of cell migration (Fig. 1A). In the second stage (step 2), a ring was placed onto the marked circumference inside each well. Based on their size differences, the different EC lines were seeded inside the rings at densities of 60,000 (4725 cells/mm2), 50,000 (3940 cells/mm2), and 20,000 (1575 cells/mm2) for RAOECs, RAMECs, and HAECs, respectively. These densities were optimal for the formation of an instantaneous circular monolayer inside the rings. The EC suspensions were carefully applied into the center in a drop of 40 µl of GM and incubated at 37°C and 5% CO2 for 1 h (RAMECs) or 2.5 h (RAOECs and HAECs). A 5-mm glass bead (Fisher Scientific Co.) was placed on top of the ring (Fig. 1A) to provide stability and firm contact to the bottom of the well. In the next stage (step 3), the rings were lifted, the cell monolayers were washed twice with prewarmed PBS (with calcium and magnesium), and the experiment was initiated by the addition of 2 ml of experimental medium (EM). EM is identical in composition to GM without rhbFGF supplementation. By excluding this particular growth factor, we were able to generate accurate growth factor dose-response effects and also to use rhbFGF as a positive control.
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Data Acquisition and Evaluation. Unless otherwise stated, all experiments were terminated after 3 days. The monolayers were photographed at time 0 (Fig. 1B) before incubation with the growth factors and drugs and after 1 and 3 days (Fig. 1C). Monolayers were inspected on an inverted Nikon contrast microscope (Eclipse TE-2000-U; Nikon, Melville, NY) using a 2x long-working distance objective. All images were acquired digitally using a Hamamatsu black-and-white high-resolution camera and analyzed using Northern Eclipse software (Empix Imaging Inc., Mississauga, ON, Canada). In each circular monolayer, we separately photographed the four quadrants of the circle (Fig. 1, B and C). The quadrants were marked in the center of the tissue culture plate carefully preserving the 0, 90, 180, and 270° direction. The photographs were analyzed by superimposing a software generated radial grid (F. Dietrich and P. I. Lelkes, manuscript in preparation) along the circular cell monolayer (Fig. 1, B and C) to determine the migration of the cells at the front of the circular monolayer. Migration distance was defined as the difference between the cell monolayer fronts from the center of the ring measured at 3 days (Fig. 1Cb) compared with the cell monolayer front at 0 time (Fig. 1Ca). The mean ± S.D. of the migration of the cell monolayer is presented in micrometer or as percentage of migration with respect to control (untreated cells).
Statistical Analyses. In general, 10 radii were measured in each quadrant (n1 = 10), taking into account at least three (the most regular cell front monolayers quadrant) of the four quadrants (n2 = 3) to produce in each ring experiment, 30 measurements. A duplicate set of rings was used for a given experimental condition (n3 = 2), generating 60 total measurements of each condition. Each OM experiment was repeated at least three times. The OM experiments described in Fig. 4 represent mean ± S.D. data from up to 10 different experiments (n4 = 3–10). Statistical significance was determined using Student's t test and/or one-way analysis of variance (ANOVA) followed by Bonferroni's post-tests or Newman-Keuls multiple comparison tests. In general, we considered the difference between groups to be significant for p < 0.01, with certain exceptions listed in the text, where we accepted p < 0.05 as statistically significant.
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To test the effect of growth factors on HAEC migration with minimal contribution of proliferation and because NGF effect on migration was maximal at 2% FBS (Fig. 4, inset), all further migration experiments were performed in 2% FBS. As seen in Fig. 3, spontaneous migration varied between the three endothelial cell lines, with RAMEC > HAEC > RAOEC. One possible reason for these differences in the migration of diverse ECs may be the distinct levels of endogenous growth factors released into the medium (Hannan et al., 1988). Given the importance of HAECs as a model system for the human vasculature, most of the subsequent experiments were carried out with HAECs.
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NGF-Induced Endothelial Cell Migration. To further validate the OM assay, we characterized the effects of two known angiogenic factors, rhVEGF and rhbFGF. As seen in Fig. 4, both these angiogenic growth factors significantly stimulated migration of HAEC cells by 1.4-(1 day) and 1.6-fold (3 day) for rhbFGF and 1.2-(1 day) and 1.3-fold (3 day) for rhVEGF, respectively. Furthermore, the data in Fig. 4 also indicate that, among the different NGFs, vNGF had the strongest effect by stimulating HAEC migration by 1.1- and 1.3-fold on days 1 and 3, respectively. By contrast, identical doses of mNGF and rhNGF had a weak (3–5%) stimulatory effect, which became statistically significant after 3 days (Fig. 4). Viper NGF stimulated migration of RAMECs in a serum-dependent manner (Fig. 4, inset). Upon 3 days of treatment with growth factors, migration of RAOECs was moderately enhanced by vNGF (13 ± 4%), whereas exposure to rhbFGF and rhVEGF resulted in an 80 ± 4 and 20 ± 3% enhancement of migration, respectively (data not shown). Together, these data provide evidence that NGFs enhances migration in all of the three endothelial cells investigated.
To further quantitatively characterize growth factor stimulation of HAEC migration, we established dose-response curves for rhVEGF, rhbFGF, and vNGF (Fig. 5). Apparent EC50 values were calculated from the log linear part of the dose-response curves generating values of 0.4, 0.5, and 0.6 ng/ml for rhVEGF, vNGF, and rhbFGF, respectively. These EC50 values indicate a similar high potency among these three growth factors in stimulating HAEC migration. However, a comparison of the maximal effects (Fig. 5) suggests that vNGF similar to rhVEGF was less efficacious than rhbFGF.
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), and SU-5416, which is a selective inhibitor of Flk-1/KDR, one of the VEGF receptors (Mendel et al., 2000a). As seen in Fig. 6, K252a at a nontoxic, selective concentration of 30 nM completely blocked vNGF-induced stimulation of HAEC migration at both 1 and 3 days of treatment, indicating that this effect of vNGF is TrkA-mediated. We also noticed that K252a inhibited the spontaneous migration of HAEC cells by 20 ± 1%, suggesting either a constitutive release of NGF by the cells or a nonspecific effect of K252a on other cellular protein kinases involved in regulation of migration. When HAECs were treated with a concentration of 1 µM SU-5416, which is clinically relevant (Mendel et al., 2000b), the drug almost completely inhibited rhVEGF-induced cell migration (Fig. 7A). From dose-response experiments in which HAECs were exposed for 3 days to 10 ng/ml rhVEGF in the presence and absence of various concentrations of SU-5416, the apparent IC50 of SU-5416 was calculated to be 0.9 µM (Fig. 7B). At this concentration, SU-5416 exerted a significant nonspecific inhibitory effect on vNGF-induced HAEC migration (data not shown). However, at a nontoxic concentration of 0.25 µM (IC5–10%), we observed a significant inhibition of SU-5416 on rhVEGF-induced but not vNGF-induced HAEC migration (Table 2).
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Potentiation of NGF-Induced HAEC Migration by rhVEGF and rhbFGF. Our data so far described independent stimulatory activities of three different angiogenic factors: vNGF, rhVEGF, and rhbFGF. We investigated possible physiologically relevant interactions among different combinations of these growth factors at concentrations that, according to the dose response curves presented in Fig. 5, individually generated up to 10% stimulation of total HAEC migration. By comparison with the stimulatory effects of each individual growth factor alone, exposure to either the three pairs of growth factors or to a combination of all three angiogenic factors yielded significant additive enhancement of HAEC migration (Table 3).
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For this study, we adapted and optimized a recently described omnidirectional migration assay (Dixit et al., 2001). An important validation of our OM assay is the reproducible stimulation of the migratory response of diverse ECs upon treatment with bFGF and VEGF, which has previously been documented in a variety of two- and three-dimensional migration assays (Yoshida et al., 1996; Vernon and Sage, 1999). In our hands, the EC50 values for all growth factors were in the range of 0.4 to 0.6 ng/ml, indicating that our OM assay is very sensitive, probably because we used a radial migration measurement approach and not a surface area approach (Dixit et al., 2001). These values are in line with the Kd values obtained for bFGF and VEGF binding to their respective receptors (Neufeld and Gospodarowicz, 1985; Soker et al., 1996). By comparison, in previous studies using these growth factors, concentrations between 1 and 10 ng/ml were used to measure the effects of VEGF and bFGF on the migration of cultured ECs (Yoshida et al., 1996; Ghosh et al., 2002).
A comparison between the maximal effects of these growth factors on HAEC migration indicates that rhbFGF is more efficacious than rhVEGF (Fig. 5). This finding is consistent with other migration studies performed with bovine aortic endothelial cells in which bFGF and VEGF induced 155 and 135% stimulation, respectively, of EC migration in a "wound model"-type migration assay (Ghosh et al., 2002).
A known difficulty in assessing cell migration in most in vitro assays is that cell proliferation may contribute in part to the measured migration (Cai et al., 2000). Preliminary data indicate that at day 3 of vNGF-stimulated migration, only 6 ± 1% HAECs were proliferating at the front of the circular monolayer (unpublished results). Thus, the contribution of proliferation to HAEC migration in the present OM assay may be relatively small and requires further investigation.
In this study, we compared the effects of several NGF agonists on HAEC cell migration. Mouse and human recombinant NGFs generated a weak yet statistically significant stimulatory signal. By contrast, viper NGF stimulated the migration of HAECs with a potency and an efficacy similar to that of rhVEGF. The higher potency of vNGF by comparison to the other NGFs may be attributed either to the Asn-21 glycosylation found only in vNGF (Katzir et al., 2003) but not in mouse and human recombinant NGF (Hayashi et al., 1996) or to an increased affinity toward TrkA receptors due to primary sequence changes compared with the other NGFs. It is well known that glycosylation of NGF and FGF results in an increased stability, probably causing more efficient and/or prolonged receptor stimulation (Delli-Bovi et al., 1988; Murphy et al., 1989).
Interestingly, the order of potency by which the various NGF analogs stimulated HAEC migration (vNGF >> mNGF > rhNGF) is opposite to the order by which these agonists induce neurite outgrowth (rhNGF > mNGF > vNGF) in PC12 cells overexpressing neuronal human recombinant TrkA receptors (Katzir et al., 2003). This observation suggests that the TrkA receptor in HAECs may be of another subtype than human neuronal TrkA. Together, we propose that vNGF may serve as an important tool to study angiogenic effects of NGF in ECs in vitro and in vivo (Lazarovici et al., 2005).
Two receptor antagonists, K252a and SU-5416, are important tools for probing the specificity and selectivity of NGF- and VEGF-induced HAEC migration, respectively. At the low concentrations used in our studies, both K252a and SU-5416 are highly specific antagonists of the cognizant high-affinity receptors for NGF and VEGF, TrkA, and Flk-1/KDR, respectively. Under these conditions, we demonstrated that NGF-induced HAEC migration was not blocked by SU-5416, excluding the possibility that NGF effect is mediated directly by autocrine release by VEGF. This latter possibility has been favored in a recent in vivo study (Manni et al., 2005).
NGF-induced migration of aortic ECs confirms and extends recent similar observations in porcine aortic ECs (Rahbek. et al., 2005) and human choroidal ECs, but interestingly, not in human retinal ECs (Steinle and Granger, 2003). NGF-induced migration of ECs is in line with previous reports on the angiogenic effects of mouse and human recombinant NGF in vitro and in vivo. NGF has been shown to stimulate proliferation of HUVECs (Cantarella et al., 2002), human choroidal endothelial cells (Steinle and Granger, 2003), human dermal microvascular endothelial cells (Raychaudhuri et al., 2001), and rat brain endothelial cells (Moser et al., 2004). NGF also promoted survival of mice aortic endothelial cells (Tanaka et al., 2004) and increased neoangiogenesis in the chick chorioallantoic membrane (Cantarella et al., 2002). In view of the well known phenomenon of EC heterogeneity (Lelkes et al., 1996), future studies will focus on the similarities and differences in angiogenic response among ECs of different tissue origins in response to NGF.
Generally, in the cardiovascular system, a number of different angiogenic factors are operative concomitantly. Hence, we studied the possible relationship among VEGF, bFGF, and NGF. As described in Table 3, the three growth factors additively stimulated EC migration, suggesting similarities in their mechanism of action. Previous studies indicated the presence of Flk-1/KDR (Endo et al., 2003), FGF receptor (Motamed et al., 2003), and TrkA (Rahbek et al., 2005) in aortic endothelial cells. As soon as one of the above-mentioned receptors is stimulated, the tyrosine phosphorylation of its cellular substrates initiates the signaling pathways that result in cell migration. Hence, stimulation of yet another tyrosine kinase-activating receptor may induce only a weakly additive effect. A synergistic effect would have required mobilization of a completely different signal transduction pathway. We propose that the potentiation of two-dimensional HAEC migration by the above-mentioned growth factors is analogous to similar effects seen in vitro in three-dimensional models (Pepper et al., 1992) as well as in in vivo experiments (Asahara et al., 1995).
The signaling pathways of NGF-induced migration or chemotaxis of endothelial and nonendothelial cells involve phosphatidylinositol 3-kinase, extracellular signal-regulated kinase 1 and 2, Src, Rho GTPase Rac 1, cdc42 kinase, and paxillin (Escalante et al., 2000; Steinle and Granger, 2003; Ho et al., 2005; Rahbek et al., 2005). An important issue to be elucidated is the role of each of the two NGF receptors, viz., TrkA and p75NTR in NGF-induced HAEC migration. p75 NTR modulates the migration of primary melanoma (Shonukan et al., 2003) and Schwann cells (Yamauchi et al., 2004). Our preliminary DNA array data indicate that HAECs express p75 NTR; inhibition of NGF-induced HAEC migration by K252 implicates the presence of trkA, in line with the findings by Rahbek et al. (2005). The precise role and signaling pathways of both NGF receptors are currently under investigation.
In conclusion, we propose that NGF-induced migration of ECs may be paradigmatic for the widespread cross-talk between the nervous and cardiovascular systems (Lazarovici et al., 2005). For example, sympathetic denervation results in significant blood vessel growth (Torry et al., 1991) that may be related to an increased NGF production by aortic tissue (Ueyama et al., 1991). It is tempting to speculate that NGF is necessary to induce proliferation and/or migration of ECs, which in turn will lead to the repair of cardiovascular tissue. Our observation of NGF-induced migration of cultured aortic cells may be relevant also for angiogenic processes in vivo.
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Footnotes |
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ABBREVIATIONS: EC, endothelial cell; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; NGF, nerve growth factor; HAEC, human aortic endothelial cell; RAOEC, rat aortic endothelial cell; RAMEC, rat adrenal medullary endothelial cell; OM, omnidirectional migration; FGF, fibroblast growth factor; rhVEGF, human recombinant vascular endothelial growth factor; rhbFGF, recombinant human basic fibroblast growth factor; Flk-1/KDR, subtype of VEGF receptors (VEGFR2); SU-5416, 3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one; K252a, (8R*,9S*,11S*)-(|)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; rhNGF, recombinant human nerve growth factor; mNGF, mouse nerve growth factor; vNGF, viper nerve growth factor; GM, growth medium; FBS, fetal bovine serum; EM, experimental medium; ANOVA, analysis of variance.
Address correspondence to: Dr. Peter I. Lelkes, Calhoun Chair Professor of Cellular Tissue Engineering, School of Biomedical Engineering, Science and Health Systems, Drexel University, 3141 Chestnut St., Philadelphia, PA 19104. E-mail: pil22{at}drexel.edu
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, 
, p < 0.01, by comparison between the different endothelial cells, one to the other at each individual time point using Student's t test. Single symbols represent comparisons between the various species; double symbols represent comparison of the migration distances of the same species measure at days 1 and 3, respectively.
, vNGF; 
, rhbFGF. Control migration was 335 ± 19 µm (100%).
, p < 0.01 compared with control;