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1 Department of Cell Biology and Anatomy, Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology; 2 Department of Pathology, Carmel Medical Center; and 3 Department of Biotechnology and Food Engineering, Technion-Institute of Technology, Haifa, Israel
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ABSTRACT |
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INTRODUCTION |
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10-fold lower than its affinity to np2. However, s3f is unable to repulse neuronal growth cones expressing np1, indicating that s3f cannot transduce biological signals via this receptor (8)
. Other class-3 semaphorins also bind to np1, np2, or both receptors, but these semaphorins have been less extensively studied (9)
. The 165 amino acids form of vascular endothelial growth factor (VEGF165) also binds to both neuropilins. In contrast, VEGF145 is able to bind to np2, but not np1, whereas VEGF121 does not usually bind to neuropilins (10, 11, 12) . VEGF165 binding to np1 enhances VEGF165 signaling mediated by the VEGF receptor (VEGFR)-2 (10) . Additional factors belonging to the VEGF family, such as placenta growth factor, VEGF-B, and VEGF-C, are also able to bind to specific neuropilins (13, 14, 15) . In early chick and mouse embryos, np1 is mainly expressed in arteries, whereas np2 is expressed in veins. At later stages of development, np2 expression appears in lymphatic vessels, and the expression in veins is down-regulated (16, 17, 18) . Gene-targeted mice lacking functional np1 display severe cardiovascular and neuronal abnormalities (19) . In contrast, np2 gene-targeted mice are viable. They display neuronal abnormalities, but the cardiovascular system is normal, except for some abnormalities in peripheral lymph vessels (18) . However, mice in which both neuropilin genes were inactivated display cardiovascular defects that are much more acute than those generated by the inactivation of a single neuropilin, thereby confirming a role for both neuropilins in vasculogenesis and developmental angiogenesis (20) .
These studies indicate that semaphorins may perhaps be able to function as modulators of vasculogenesis, angiogenesis, and possibly lymphangiogenesis. Indeed, s3a was found to inhibit capillary sprouting in an in vitro assay of angiogenesis and VEGF165-induced migration of endothelial cells. s3a inhibits the binding of VEGF165 to np1, and it was therefore postulated that the s3a inhibits angiogenesis by inhibiting the potentiating effect that VEGF165 binding to np1 confers on VEGFR-2-mediated signal transduction (10 , 21) . In addition, it was recently shown that s3a can inhibit the adhesion of endothelial cells to extracellular matrix constituents, such as fibronectin, and inhibit developmental angiogenesis in chick embryo (22 , 23) . These effects of s3a may or may not be linked to its ability to induce apoptosis (24) . Surprisingly, there were no gross effects on vascular system organization in mice that were gene targeted for s3a, except for heart malformations (25) .
In contrast to s3a, which competes with VEGF165 for binding to np1, s3f does not compete with VEGF165 for binding to np2, although both factors bind to np2 (12) . The vascular systems of mice that lack functional s3f genes appear relatively normal, and the mice were viable (26) , indicating that on its own, s3f does not fulfill a critical function during the organization of the embryonic vascular system. s3f had been originally isolated as a tumor suppressor of small cell lung carcinoma (3 , 5) and had been shown to affect the proliferation and migration of tumor cells that express s3f receptors (27) . In the present study, we have examined the effects of s3f on the proliferation of human umbilical vein derived endothelial cells (HUVECs). The proliferation and survival of these cells in vitro depend on the addition of exogenous angiogenic growth factors, such as VEGF or basic fibroblast growth factor (bFGF). s3f inhibited the mitogenic effects of VEGF165 and bFGF in these cells, indicating that s3f may be able to function as an inhibitor of angiogenesis. Indeed, we show that s3f inhibits VEGF165 and bFGF-induced angiogenesis in vivo. Furthermore, s3f also inhibited the formation of tumors derived from human embryonic kidney (HEK)293 cells, although s3f was not able to inhibit the proliferation of these cells in cell culture. The development of the few tumors that did develop from s3f-expressing HEK293 cells was retarded, and the tumors contained significantly lower densities of blood vessels, indicating that s3f functions as an inhibitor of tumor angiogenesis.
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MATERIALS AND METHODS |
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Production and Purification of s3f.
The cDNA encoding the longer 786 amino acids splice form of s3f (s3f-l) or the shorter 755 amino acids splice form of s3f (4)
was ligated in-frame into the pcDNA3.1/myc-His expression plasmid to generate expression vectors encoding myc and 6xHis epitope-tagged s3f splice forms. HEK293 and BHK-21 cells were transfected with these expression vectors using Lipofectamine and stable s3f- and s3f-l-expressing clones isolated using G418 (0.5 mg/ml) selection. To purify s3f, HEK293 cells transfected with the s3f expression vector or empty expression vector were cultured to 90% confluence. The medium was exchanged to serum-free DMEM. Sodium butyrate (5 mM) was added after 24 h to the control and s3f-expressing cells, and the medium was collected after an additional 24 h. HEPES buffer (10 mM, pH 7.3) and protease inhibitors (0.2 mg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin and aprotinin, and 1 mM EDTA) were then added. The medium was incubated overnight at 4°C with wheat-germ agglutinin agarose (1 ml of beads/0.5 liter of medium). The column was washed twice with PBS, and bound s3f was eluted using 0.5 M N-acetylglucosamine in PBS. The s3f peak in the eluant was dialyzed overnight at 4°C against PBS in bags with a cutoff of Mr 50,000. The dialyzed s3f was loaded onto an S-Sepharose column (0.5 ml of bed volume) pre-equilibrated with wash buffer [20 mM NaHPO4 (pH 7.2) and 1 mM EDTA]. The column was washed twice with 5 ml of wash buffer, once with 2 ml of wash buffer containing 0.1 M NaCl, and eluted with wash buffer containing 0.5 M NaCl (Fig. 1B)
. s3f containing fractions were pooled and frozen in liquid nitrogen.
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Cell Contraction.
Cells were incubated for 30 min at 37°C with 5 µg/ml DiAsp in serum-free medium. The cells were washed with HBSS and incubated in CHO-S-SFM II medium containing s3f or s3f-l or with a control-conditioned medium from empty vector-transfected cells for 1 h at 37°C, after which the medium was aspirated. Alternatively, the experiment was done using purified s3f and respective control fractions. The cells were fixed with 4% paraformaldehyde and photographed using an inverted fluorescent microscope. PhotoShop in conjunction with the scion image program were used to measure the circumference of 80 randomly selected cells from several representative fields from each experiment.
ERK-1/2 Phosphorylation.
HUVECs were seeded at a concentration of 5 x 105 cells in 6-well gelatinized dishes in growth medium containing 10% FCS. The cells were allowed to attach and incubated 16 h in a humidified incubator at 37°C. The experiment was initiated by the addition of increasing concentrations of s3f or corresponding volumes of a corresponding control fraction purified similarly from control cells transfected with empty expression vector. After 10 min at room temperature, bFGF (2 ng/ml) or VEGF165 (10 ng/ml) was added or not added to dishes. The experiment was terminated after 10 more min by a wash with ice cold PBS. The cells were lysed with 0.03 ml of lysis buffer containing 50 mM HEPES (pH 7.4), 4 mM EDTA, 1% Triton X-100, 0.5 mg/ml Na3VO4, 4.5 mg/ml Na4P2O7, and fresh protease inhibitors (0.2 mg/ml phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin). The cells were scraped off, and nonsoluble debris was removed by centrifugation at 4°C. Aliquots of supernatant containing 10 µg of protein were loaded on an SDS-PAGE gel, blotted onto a nitrocellulose filter, and probed with an antibody directed against phosphorylated ERK-1/2. The blot was then stripped and reprobed with an antibody directed against ERK-2 (total ERK). Quantification of band intensity was performed using a phosphorimager (Image Master, Amersham), and the ratio between phosphorylated ERK and total ERK was determined using the Tina program.
Alginate Microencapsulation Angiogenesis Assay.
BHK-21 cells transfected with empty expression vector, BHK-21 cells expressing either s3f-l or s3f, or BHK-21 cells expressing VEGF165 (30)
were encapsulated in polylysine-coated microspheres composed of Ca+2-alginate as described previously (29
, 31)
. To evaluate the concentration and viability of the encapsulated cells, capsules containing cells were incubated with 5 mg/ml 3-(4
,5)
-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide for 5 h in 37°C, washed three times with 10 mM HEPES (pH 7.3), and incubated for 24 h in isopropanol containing 0.4% HCl at room temperature. Absorbance at 570 nm was then measured. After a week in culture, conditioned medium was assayed for recombinant s3f or s3f-l (using anti-myc epitope antibodies) or VEGF165 (using anti-VEGF165 antibodies; Ref. 32
). Capsules containing VEGF165-producing cells were mixed with capsules containing s3f- or s3f-l-expressing cells or control cells and injected s.c. as described previously (29)
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Matrigel Plug Angiogenesis Assay.
Matrigel containing 80 ng/ml bFGF and either s3f (33 nM) or a corresponding volume from the corresponding control fraction was prepared and used for the Matrigel plug angiogenesis assays as described (33)
. Matrigel (0.6 ml/animal) was injected s.c. into female immune-deficient mice (BALB/c nu/nu). The animals were sacrificed, and the Matrigel plugs were removed and photographed after 7 days. Plugs were embedded in paraffin blocks and sectioned. Sections (5-µm thick) were reacted with an antibody directed against CD-31 as described (28)
. The sections were counterstained with hematoxilin, and CD-31-positive blood vessels that have invaded the Matrigel were visualized. The microvessel density of CD-31-positive vessels in fields of equal area was measured. Seven fields from three control and s3f-treated plugs were quantified. Microvessel density was measured using the hot spot method according to which the most vascularized fields in different samples are assessed for vascular density (34)
. The quantification of the blood vessel density was performed using the ImagePro Plus version 4.5 software (MediaCybernetics, Silver Spring, MD). This method was also used to determine the density of blood vessels in sections derived from tumors in a similar fashion. Vascular density was expressed as a percentage of area relative to the area of the microscopic field. All animal experiments were approved by the Technion ethics commission.
Cross-Linking of 125I-bFGF to Cells.
BHK-21 or PAE cells were grown to confluence in gelatin-coated, 5-cm dishes. The production of recombinant bFGF from bacteria-expressing recombinant bFGF, iodination of the bFGF, and the binding and cross-linking of 3 ng/ml 125I-bFGF to cells was done as described previously (35
, 36)
in the presence or absence of 15 nM s3f.
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RESULTS |
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. PAE cells expressing recombinant plexin-A1 and np2 contract strongly and rapidly in response to s3f (compare Fig. 1, C with D
). In contrast, PAE cells expressing only np2 or parental PAE cells did not contract in response to s3f (data not shown). HUVECs express np1, np2, plexin-A1, and plexin-A2 as shown by reverse PCR (Fig. 1B)
. However, the contraction of HUVEC in response to s3f is relatively weak (compare Fig. 1, E with F
), although measurements of cell circumference using the scion image program indicate that this small contraction is statistically significant as determined using two-tailed unequal Student’s t test (P < 0.01; data not shown).
S3f Inhibits VEGF165 and bFGF-Induced Survival and Proliferation of HUVECs.
To find out if s3f affects the survival of HUVECs, we cultured the cells in medium containing 1% serum. Under these conditions, VEGF165 acts as a survival factor. The addition of 
10 nM s3f (4)
to the medium inhibited significantly the survival-inducing effect of VEGF165 (Fig. 2A)
. Similar results were obtained using human saphenous vein-derived endothelial cells (data not shown). Inactivation of s3f at 80°C for 5 min abolished the inhibitory effect of s3f (data not shown). The inhibitory effect of s3f was also evident in the presence of serum because s3f also inhibited strongly VEGF165-induced proliferation in 10% serum (Fig. 2B)
. The inhibition was specific to s3f because corresponding column fractions purified similarly from conditioned medium of HEK-293 cells transfected with empty expression vector (control fractions) failed to inhibit the effects of VEGF165 (Fig. 2, A
and B, controls).
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, indicating that at least part of the inhibitory effect of s3f is generated by a mechanism that does not rely on the inhibition of the interaction of proangiogenic factors such as bFGF with bFGF receptors. Half maximal inhibition of VEGF165 activity was observed at 1.5 nM s3f (Fig. 2A)
, whereas half maximal inhibition of bFGF activity was observed at 2 nM s3f (Fig. 2C)
. These measurements were obtained in the presence of saturating concentrations of VEGF165 and bFGF and thus do not represent accurate IC50 measurements. However, they do show that the range of s3f concentrations required for the inhibition of VEGF and bFGF-induced cell proliferation is similar. Increasing the concentration of bFGF 50 ng/ml did not reverse significantly the inhibitory effect produced by 5 nM s3f, although a minor reversal of 10% could be seen (Fig. 2D)
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S3f Inhibits VEGF and bFGF-Induced Phosphorylation of ERK-1/2.
The ERK-1 and ERK-2 mitogen-activated protein kinases are positioned near the end of the ras signaling cascade and phosphorylated in response to VEGF and bFGF in HUVECs (39
, 40)
. Purified s3f, but not control fractions, inhibited the VEGF165-induced phosphorylation of ERK-1/2 (compare Fig. 3A
, Lanes 3 with 4). Similar inhibition was seen when the effects of increasing concentrations of s3f on bFGF-induced activation of ERK-1/2 were compared with the effects of correspondingly increasing volumes of a control fraction, although the inhibition was not as complete as inhibition of VEGF activity (Fig. 3B)
. The ratio between phosphorylated ERK-1/2 and the amount of total ERK was quantified for each of the points in this experiment, and the results are shown in Fig. 3C
. The inhibition experiments were repeated six times with similar results, and when the inhibition was analyzed statistically (at 5 nM s3f), it was found to be highly significant (P < 0.01 using Student’s t test). Because the induction of ERK-1/2 phosphorylation and inhibition occur within 10 min, these results indicate that inhibition of receptor up-regulation is probably not part of the mechanism by which s3f inhibits the activities of VEGF and bFGF.
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, indicating that the mitogenic activity of bFGF is not mediated indirectly by VEGF and that s3f does not inhibit the mitogenic effects of bFGF by using a mechanism that involves competition for shared receptors.
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. However, inclusion of capsules containing cells producing s3f-l inhibited VEGF165-induced angiogenesis (Fig. 5A, e–h)
. Similar results were obtained in an experiment in which BHK-21 cells expressing s3f were used (data not shown). Implantation of capsules containing s3f-expressing cells or parental BHK-21 cells on their own did not induce angiogenesis (data not shown).
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). The s3f-induced inhibition of angiogenesis was sensitive to the ratio of the VEGF165 and s3f-producing cells in the alginate bead mixtures. When the number of capsules containing VEGF165-producing cells was increased 5-fold without a corresponding change in the number of the s3f-expressing cells, we could no longer see such efficient inhibition of angiogenesis, although even under these conditions, a small effect was evident (data not shown).
To determine whether s3f is also able to inhibit the angiogenic activity of bFGF, we used the Matrigel plug angiogenesis assay (33)
. bFGF is not secreted efficiently from producing cells because it lacks a signal sequence for secretion, rendering the alginate cell encapsulation system unsuitable for the determination of bFGF-induced angiogenesis. It can be seen that s3f inhibited efficiently bFGF-induced growth of blood vessels into the Matrigel plugs, indicating that s3f can inhibit bFGF-induced angiogenesis, as well as VEGF165-induced angiogenesis (Fig. 5B, a–c)
. In contrast, control fractions purified by an identical procedure from conditioned medium of empty vector-transfected HEK-293 cells did not inhibit bFGF-induced angiogenesis (Fig. 5B, a–c)
. This conclusion was verified by determining blood vessel densities in CD-31-labeled histological sections derived from control and s3f-containing Matrigel plugs, such as those shown (Fig. 5B
, g and h, black arrows). Using the hot spot method (34)
, we have found that bFGF and control-containing plugs had a 6-fold higher blood vessel density as compared with plugs containing bFGF and s3f (Fig. 5D)
. It should be noted that bFGF stimulated the migration of additional cell types into the Matrigel plugs (note the density of cells that do not express CD-31 in Fig. 5h
) and that s3f also inhibited partially the migration of nonendothelial cell types. However, we have not determined which other cell types were inhibited by s3f.
Expression of s3f in HEK-293 Cells Inhibits Tumor Formation but not Cell Proliferation.
s3f was identified as a suppressor of small cell lung carcinoma (3
, 5)
. HEK293 cells transformed by adenovirus 5 (45)
form slowly developing s.c. tumors in nude mice with an incidence of 50% (46)
. We have expressed in HEK293 cells the two s3f splice forms (4)
. The proliferation rate of cells expressing s3f-l (data not shown) or s3f was compared with the rate of proliferation of empty vector-transfected cells. The rate of proliferation of the s3f (Fig. 6A)
or s3f-l-expressing cells (data not shown) did not differ significantly from the proliferation rate of control cells, indicating that s3f does not inhibit the proliferation of these cells. It is possible that the concentrations of s3f do not build up quickly in cell culture because the initial number of the cells in such experiments is low. We have also conducted similar experiments in which we have added to the control and s3f-producing cells exogenous s3f. However, exogenous s3f did not significantly affect the proliferation rate of the control or s3f-expressing cells (Fig. 6B)
.
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. Furthermore, the three tumors that developed from cells expressing s3f-l developed at a much slower rate as compared with tumors derived from empty vector-transfected cells (the average weight of tumors after 7 weeks was 0.3 gram, whereas the average weight of control tumors was 2 grams). Theses observations suggest that s3f inhibits the development of HEK293-derived tumors by inhibiting tumor angiogenesis.
These impressions were confirmed when the density of blood vessels was compared between the control tumors and the three tumors that did develop from the s3f-l-expressing HEK293 cells. The density of the blood vessels in the tumors that developed from the s3f-expressing cells was indeed significantly lower than the density of blood vessels in control tumors (Fig. 6D)
, indicating that s3f can function as an inhibitor of tumor angiogenesis.
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DISCUSSION |
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It was reported that the angiogenic activity of bFGF is mediated in part by an up-regulation of VEGF (41) and, furthermore, that bFGF can enhance the synthesis of VEGFR-2 receptors in endothelial cells (47) . It is thus possible that s3f could inhibit the activity of bFGF, at least partially, by inhibiting the interaction of VEGF produced in response to bFGF with a receptor such as np1, although the affinity of np1 toward s3f is significantly lower than the affinity of the np2 receptor (8) . However, the involvement of such a VEGF/VEGFR-dependent mechanism is ruled out because sflt, a potent inhibitor of VEGF function (48) , does not inhibit bFGF-induced proliferation of HUVEC and because s3f can inhibit the mitogenic effects of bFGF, even in the presence of sflt. Therefore, it is unlikely that VEGF produced in response to bFGF plays a role in the mitogenic response to bFGF in vitro. In addition, we have shown that s3f inhibits bFGF and VEGF-induced phosphorylation of ERK-1/2 in HUVECs. Because the phosphorylation of ERK-1/2 in response to growth factors is a very rapid response, and up-regulation of receptors, as well as synthesis of a growth factor such as VEGF, is a much slower process, it follows that the mechanism by which s3f inhibits the activities of bFGF and VEGF in HUVECs is not likely to depend on the up-regulation of VEGF expression.
The evidence gathered in this set of experiments therefore suggests that s3f inhibits bFGF-induced proliferation of HUVECs by using a mechanism that does not rely on the inhibition of the binding of bFGF to its receptors. This mechanism probably uses instead a mechanism that requires active signal transduction, most likely via the np2 and type A plexin receptors. Np1 receptors, although able to bind s3f with low affinity, do not transduce s3f signals (6 , 8) and are unlikely to transduce antiproliferative s3f signals, although this will need to be verified experimentally in the future. These findings imply that s3f could function in vivo as a localized inhibitor of angiogenesis that may be able to inhibit the activity of a broad range of angiogenesis-inducing factors.
Proteins that inhibit the proliferation of endothelial cells in vitro are expected to inhibit angiogenesis in vivo. Indeed, by using the alginate microencapsulation and Matrigel plug angiogenesis assays, we have shown that the two splice forms of s3f inhibit significantly VEGF165, as well as bFGF-induced angiogenesis. It follows that part of the antitumorigenic properties of s3f (5) may be attributable to its antiangiogenic effects. Most tumor cells express low levels of np1 or np2 mRNA, and it is therefore difficult to determine whether the s3f inhibition of tumor growth is the result of a direct effect on the proliferation of the tumor cells or the result of an antiangiogenic effect. Our experiments with the HEK293 cells indicate that s3f can inhibit tumor growth in vivo, although s3f is not able to inhibit the proliferation of the HEK293 cells in cell culture. In conjunction with the previous experiments, this experiment argues strongly that s3f can inhibit tumor development by inhibiting tumor angiogenesis. Furthermore, the few tumors that did develop from cells expressing s3f developed much slower and had a significantly lower density of blood vessels, indicating that impairment of angiogenesis contributed significantly to the retardation of their growth. However, there are tumorigenic cell types, such as the GLC45 small cell lung carcinoma cells that were reported to form tumors efficiently in nude mice, even in the presence of s3f (5) . Because tumor development depends on the induction of tumor angiogenesis (49) , why then is tumor development not inhibited by s3f in the case of the GLC45 cells? The answer may perhaps reside in the balance between inducers and inhibitors of angiogenesis. Our experiments indicate that the in vivo inhibitory effects of s3f can be partially overcome by increasing the ratio between VEGF165 and s3f-producing cells in favor of VEGF165-producing cells. It is thus possible that GLC45 cells produce high concentrations of angiogenic factors, thereby overcoming the antiangiogenic effects of s3f.
It is not known whether s3f plays a role in the regulation of physiological angiogenesis. Mice lacking functional np2 genes develop almost normally as far as the cardiovascular system is concerned, and excessive growth of blood vessels or abnormal organization of the vascular system was not noted (18 , 50 , 51) . Likewise, mice deficient in the expression of s3f do not display gross abnormalities in their cardiovascular system (26) . However, it is possible that s3f plays a role in the regulation of localized angiogenesis and especially in pathological angiogenesis. Similar observations were recently reported in relation to the angiogenic factor placenta growth factor, which has only a minimal role, if any, in developmental angiogenesis but plays a major role in pathological angiogenesis (52) .
The mechanism by which s3f inhibits angiogenesis is still unclear. It is most likely that the effects are the result of an interaction with np2 receptors, which had been convincingly demonstrated to transduce s3f signals. However, it is possible that not all of the effects of s3f are mediated by np2. Although thus far signaling of s3f via np1 receptors could not be detected (6 , 8) , it is possible that the relatively weak interaction of s3f with np1 can nevertheless activate alternative signaling pathways. These questions will need to be addressed in the future so as to better understand the mechanisms by which s3f modulates angiogenesis and to find out whether s3f functions as a physiological modulator of angiogenesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: O. Kessler and N. Shraga-Heled contributed equally to this work.
Requests for reprints: Gera Neufeld, Department of Cell Biology and Anatomy, Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, 31096, Israel. Phone: 972-8523672; Fax: 972-8523947; E-mail: gera{at}tx.technion.ac.il
Received 10/ 1/03. Revised 11/25/03. Accepted 12/ 2/03.
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C. Christensen, N. Ambartsumian, G. Gilestro, B. Thomsen, P. Comoglio, L. Tamagnone, P. Guldberg, and E. Lukanidin Proteolytic Processing Converts the Repelling Signal Sema3E into an Inducer of Invasive Growth and Lung Metastasis Cancer Res., July 15, 2005; 65(14): 6167 - 6177. [Abstract] [Full Text] [PDF]
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 V. Potiron and J. Roche Class 3 Semaphorin Signaling: The End of a Dogma Sci. Signal., May 24, 2005; 2005(285): pe24 - pe24. [Abstract] [Full Text] [PDF]
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 A. Eichmann, T. Makinen, and K. Alitalo Neural guidance molecules regulate vascular remodeling and vessel navigation Genes & Dev., May 1, 2005; 19(9): 1013 - 1021. [Abstract] [Full Text] [PDF]
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 A. Sahay, C.-H. Kim, J. P. Sepkuty, E. Cho, R. L. Huganir, D. D. Ginty, and A. L. Kolodkin Secreted Semaphorins Modulate Synaptic Transmission in the Adult Hippocampus J. Neurosci., April 6, 2005; 25(14): 3613 - 3620. [Abstract] [Full Text] [PDF]
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 G. Serini and F. Bussolino Common Cues in Vascular and Axon Guidance Physiology, December 1, 2004; 19(6): 348 - 354. [Abstract] [Full Text] [PDF]
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A. R. Reynolds, L. E. Reynolds, T. E. Nagel, J. C. Lively, S. D. Robinson, D. J. Hicklin, S. C. Bodary, and K. M. Hodivala-Dilke Elevated Flk1 (Vascular Endothelial Growth Factor Receptor 2) Signaling Mediates Enhanced Angiogenesis in {beta}3-Integrin-Deficient Mice Cancer Res., December 1, 2004; 64(23): 8643 - 8650. [Abstract] [Full Text] [PDF]
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