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Dipartimento di
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Medicina Interna, and
Fisiopatologia Clinica,Università di Torino, Torino, Italy
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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subunit and
of a common transducing ß (ßc) subunit, which
does not possess an intrinsic tyrosine kinase domain (9, 10). However, activation of IL-3R is associated with the
tyrosine phosphorylation of several molecules, including the member of
the Janus family, JAK2, and the transcriptional factors, termed STATs
(11, 12, 13, 14). STAT proteins are latent cytoplasmic
transcriptional factors that, upon activation, regulate expression of
target genes (14, 15, 16, 17). Among members of the STAT family,
STAT5 consists of two highly related proteins, STAT5A and STAT5B
(18). Although STAT5 was originally identified as a
transcriptional factor activated by prolactin in the lactating mammary
gland (19), several lines of evidence indicate that STAT5
can also be activated by other cytokines (14, 15, 16, 17),
including IL-3 (18). Moreover, besides the
prolactin-inducible element (PIE) of the ß casein gene, other genes
have been identified as target for STAT5 in IL-3-stimulated cells
(18). In the present study, we evaluated the ability of hIL-3 to promote new vessel formation. We demonstrate that, in vitro, hIL-3 stimulated directional migration and tubulogenesis of both HUVEC and of murine endothelial cells, and in vivo, hIL-3 was able to induce neoangiogenesis in a murine model of Matrigel implantation. Moreover, consistent with the role of STAT5 proteins in regulating IL-3-mediated cell proliferation, we demonstrate that STAT5A and STAT5B were recruited by the IL-3R in HUVEC stimulated with hIL-3.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M199 medium (endotoxin-tested), BSA, protein A-Sepharose,
FITC-conjugated anti-rabbit IgG and anti-mouse IgG were all
from Sigma (St. Louis, MO). Bovine calf serum (endotoxin-tested) was
obtained from HyClone (Logan, UT). IMDM and RPMI were purchased from
Life Technologies (Grand Island, NY). Trypsin and gelatin were
purchased from Difco (Detroit, MI). hIL-3 was a gift from Sandoz Pharma
LTD (Basel, Switzerland). Nitrocellulose filters, HRP-conjugated
protein A, m.w. markers, [-32P]dCTP, and the
chemiluminescence reagent (ECL), were from Amersham (Braunschweig,
Germany). Poly(dIdC):poly(dIdC) was obtained from Pharmacia (Uppsala,
Sweden). Matrigel basement membrane matrix was obtained from Becton
Dickinson Labware (Bedford, MA). WEB 2170, a triazolodiazepine
(hetrazepinoic) with potent and specific PAF-receptor antagonist
activity (20), was obtained from Boehringer (Ingelheim,
Germany). Silica gel 60F254 TLC plates were obtained from Merck
(Darmstadt, Germany). µPorasil HPLC columns were provided by
Millipore (Bedford, MA). The presence of endotoxin contamination of
hIL-3 preparation was tested by the Limulus amebocyte assay
and the concentration was <0.1 ng/ml.
Antisera
Polyclonal rabbit anti-hIL-3R ßc subunit antiserum was prepared as previously described (11, 21). Rabbit polyclonal antiserum to hIL-3 (anti-hIL-3 Ab) and mAb to hIL-3 (anti-hIL-3 mAb) were from Genzyme (Cambridge, MA). Anti-mouse T cell serum, anti-L3/T4, -Ly2 mAbs, anti-CD45 Ab, anti-MAC-1 FITC-conjugated mAb, and the correspondent irrelevant isotypic IgG controls were purchased from Cedarlane (Ontario, Canada). Rabbit anti-human von Willebrand Factor, rabbit IgG and anti-rabbit IgG mAb (RG-96), FITC-conjugated goat anti-rabbit IgG, or anti-mouse IgG were obtained from Sigma. PY20 anti-phosphotyrosine Ab was obtained from Affinity Research Products (Notthingam, U.K.). Anti-STAT5A (L-20) and anti-STAT5B (G-2 and C-17) antisera were obtained from Santa Cruz Biotechnology (Heidelberg, Germany).
Cells
Endothelial cells were isolated from HUVEC within 4 h of delivery by Trypsin treatment (0.1%) and cultured in M199 with the addition of 10% bovine calf serum and 10 ng/ml of basic fibroblast growth factor (bFGF). HUVEC were characterized by morphologic criteria and positive immunofluorescence for factor VIII Ag. Contamination with blood leukocytes was assessed by immunofluorescence analysis using an anti-CD45 Ab. They were used at early passage (II-III). M-07e cells were grown as previously described (22). Polyoma T-transformed endothelial cell line (H.end), which has previously been shown to behave in vitro as normal endothelium (23, 24), was used as murine endothelium. 32Dcl3 murine hemopoietic cell line was kindly provided by Dr. G. Rovera (Wistar Institute, Philadelphia, PA).
Western blot analysis and immunoprecipitation studies
HUVEC monolayer, which had been tightly confluent for a minimum of 24 h, was washed twice with PBS and serum-starved, in endotoxin-free medium M199, containing 1% BSA, PBS (30% v/v), 0.2 mM/L sodium orthovanadate, and 1 mM/L EDTA, for 4 h at 37°C, and incubated with or without hIL-3 (20 ng/ml), PAF (10 nM/L), or WEB 2170 (3 µM/L) for the indicated time. Protein concentration of HUVEC lysates, obtained as previously described (21), was determined by the Bradford technique, and the protein content of the samples was normalized to 250 mg/sample by appropriate dilution with lysis buffer. The samples were then adsorbed by antisera coupled to protein A-Sepharose, and bound proteins were eluted and processed as previously described (21).
Preparation of nuclear extract and gel retardation assay
Nuclear extracts from untreated and hIL-3-treated M-07e cells
and untreated and hIL-3-treated HUVEC were prepared by Nonidet P-40
lysis, as described by Sadowski and Gilman (25). The
oligonucleotides used were: PIE sense 5'-G GGG GGA CTT CTT GGA ATT AAG
GGA-3' and PIE antisense 3'-G GGG TCC CTT AAT TCC AAG AAG TCC-5'
(26). The annealed oligonucleotide was labeled by filling
in the overhanging ends with Klenow fragment in the presence of
[-32P]dCTP. Gel retardation reactions were
performed as previously described (21).
In vitro endothelial cell growth assay
Proliferative activity of HUVEC was assayed by direct cell count, as previously described (8). Moreover, cell proliferation was also assessed using the colorimetric assay based on the MTT dye reaction (Boehringer Mannheim, Milan, Italy).
In vitro endothelial cell migration assay
Migration of HUVEC was performed in Boyden’s chambers. Endothelial cells that passed across the filter (8-µm pore size) after addition in the lower compartment of the chamber of the vehicle alone (saline containing 0.25% BSA), bFGF (10 ng/ml), hIL-3 (20 ng/ml), or hIL-3 + anti-hIL-3 Ab (20 ng/ml of cytokine with 20 µg/ml of Ab, preincubated for 30 min at room temperature) were counted. A possible involvement of PAF-specific receptors in endothelial cell migration was evaluated by HUVEC pretreatment with 3 µM/L of WEB 2170, a chemically unrelated specific PAF receptor antagonist (20). The specificity of the anti-hIL-3 Ab was assessed by adding in the lower compartment of the chamber bFGF + anti-hIL-3 Ab or PAF + anti-hIL-3 Ab. Chemotaxis of HUVEC in Boyden’s chambers was performed as previously described (27).
Murine angiogenesis assay
Female C57 mice were used at 6–8 wk of age. Angiogenesis was assayed as growth of blood vessels from s.c. tissue into a solid gel of basement membrane containing the test sample (28, 29). Matrigel (8.13 mg/ml), in liquid form at 4°C, was mixed with the experimental substances and injected (0.5 ml) into the abdominal s.c. tissue of mice, along the peritoneal midline. Matrigel rapidly forms a solid gel at body temperature, trapping the factors to allow slow release and prolonged exposure to surrounding tissues. The Matrigel used was extracted according to the procedure described by Taub et al. (30), that has been previously shown to efficiently deplete Matrigel of bFGF, epidermal growth factor, insulin-like growth factor, and platelet-derived growth factor (31, 32).The content of bFGF and IL-8 measured by ELISA (Quantikine, R&D System, Minneapolis MN) was <0.1 pg/ml and <3.0 pg/ml, respectively. The content of TGF-ß was 1.6 ng/ml as detected by ELISA (Quantikine). However, according to Passaniti et al. (28), TGF-ß in the Matrigel is in the latent form, which is biologically inactive. At various times, mice were subsequently killed and gels were recovered and processed for histology. Typically, the overlying skin was removed, and gels were cut out by retaining the peritoneal lining for support. Part of tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections cut at 3 mm and stained with hematoxylin and eosin were studied by light microscopy. Other sections, obtained from frozen tissue cut with a cryostat, were stained for nonspecific esterase activity (33) or processed for immunofluorescence microscopy, performed as previously described (6). Vessel area and the total Matrigel area were planimetrically assessed from stained sections, as described by Kibbey et al. (34). Considered vessels were only those structures possessing a patent lumen and containing RBC. Results were expressed as percentage ± SEM of the vessel area to the total Matrigel area. Animal procedures conformed to the Guide for Care and Use of Laboratory Resources (National Institutes of Health publication no. 93-23, revised 1985).
In vivo experimental protocol
The angiogenic effect of hIL-3 (20 ng/ml) in 0.5 ml of Matrigel was studied in the presence or in the absence of 64 U/ml heparin. In selected experiments, the effect on hIL-3-induced angiogenesis of WEB 2170, an hetrazepinoic benzodiazepin with specific PAF receptor antagonist activity (IC50 = 0.3 mM on in vitro platelet aggregation) (35) and of an anti-hIL-3 Ab was evaluated. WEB 2170 was included in the Matrigel plug (final concentration 250 ng/ml) and injected i.p. (10 mg/kg) 30 min before the s.c. injection and daily for 6 days. The anti-hIL-3 Ab, used at 20 µg/ml, was included in the Matrigel plug.
Assay and quantification of PAF
PAF bioactivity, tested after extraction (36) and purification by TLC and HPLC (37), was characterized by comparison with synthetic PAF according to the following criteria: 1) induction of platelet aggregation by a pathway independent of both ADP- and arachidonic acid/thromboxane A2-mediated pathway; 2) specificity of platelet aggregation as inferred from the inhibitory effect of PAF receptor antagonist WEB 2170 (3 µM/L); 3) TLC and HPLC chromatographic behavior and physicochemical characteristics, such as inactivation by strong bases and 5 min heating in boiling water. The methods used were previously described in detail (36).
Immunofluorescence study
To study the nuclear translocation of the activated STAT5A and STAT5B, coverslip-attached HUVEC were fixed for 5 min in 3% paraphormaldehyde in PBS (pH 7.4), containing 2% sucrose and permeabilized with HEPES-Triton X-100 buffer (20 mM/L HEPES (pH 7.4), 300 mM/L sucrose, 50 mM/L NaCl, 3 mM/L MgCl2, and 0.5% Triton X-100). STAT5A and STAT5B were detected by indirect immunofluorescence with specific anti-STAT5A and anti-STAT5B antisera (described in Antisera section) and a FITC-conjugated goat anti-rabbit IgG or anti-mouse IgG, respectively, as secondary Abs. As control, the primary antisera were substituted by preimmune rabbit serum or an irrelevant isotypic control mouse IgG.
hIL-3R analysis
The presence of the hIL-3R on murine H.end and HUVEC was evaluated by cytofluorometric analysis by assessing hIL-3 binding to the putative murine or human receptors using a technique previously described (38). hIL-3 binding to the putative receptor on the murine cell line or HUVEC was evaluated following blockade of nonspecific sites by incubation with heat-inactivated human serum for 15 min. A total of 2x106 cells, in 100 µl of staining buffer (PBS containing 2% heat-inactivated human serum and 0.1% sodium azide), was sequentially incubated for 1 h with hIL-3 (20 ng/ml), IL-1ß (10 U/ml), or a preimmune rabbit IgG for 30 min with the rabbit polyclonal anti-hIL-3 Ab, and for 30 min with FITC-conjugated goat anti-rabbit IgG. All incubations were performed at 4°C in staining buffer, and cells were washed twice between incubations. The stained cells were analyzed on a FACScan flow cytometer (Becton Dickinson).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We investigated the effect of hIL-3 on directional migration of
HUVEC. As shown in Fig. 1
, when hIL-3 was
added at a concentration of 20 ng/ml, in the lower compartment of the
Boyden’s chamber, migration of HUVEC, across the 8-µm pore-size
gelatin-coated polycarbonate filters, was observed. The hIL-3-induced
HUVEC migration was similar to that induced by 10 ng/ml of bFGF. The
motogenic activity of hIL-3 on HUVEC was also assayed in the presence
of an anti-hIL-3 Ab. The results reported in Fig. 1 demonstrated
that preincubation of hIL-3 with the anti-hIL-3 Ab significantly
decreased the hIL-3-mediated HUVEC migration, whereas no effect was
observed with the Ab alone. Moreover, pretreatment of HUVEC with the
PAF receptor antagonist, WEB 2170, significantly reduced both PAF- and
hIL-3-induced directional migration of HUVEC (Fig. 1), suggesting a
role of PAF as secondary mediator. The specificity of the
anti-hIL-3 Ab was demonstrated by its inability to block bFGF- or
PAF-induced cell migration (data not shown).
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Despite the low sequence homology between hIL-3 and murine IL-3
(29% identity) (39), Kamegai et al. (40)
have demonstrated that hIL-3 acts as proliferative and trophic factor
for central cholinergic neurons in vitro and in vivo. Therefore, we
evaluated whether hIL-3 was also able to induce directional migration
of cells from the murine endothelial cell line H.end. The results,
reported in Table I, demonstrated that
hIL-3 elicited chemotaxis of H.end and that this effect was prevented
by a blocking anti-hIL-3 Ab. We have also comparatively evaluated
binding of hIL-3 on H · end and on HUVEC. As shown in Fig. 2, both H · end (Fig. 2A)
and HUVEC (Fig. 2B) exhibited a significant hIL-3-binding
(40% H · end and 50% HUVEC) when cells were incubated with hIL-3
(thick line). No staining was observed by incubating cells with IL-1ß
(continuous line) or with preimmune rabbit IgG (dotted line). The
ability of hIL-3 to sustain the growth of the IL-3-dependent murine
hemopoietic cell line 32Dcl3 was also evaluated. Consistent with
previous reports (39), we found that hIL-3 was ineffective
on the proliferative activity of this cell line (data not shown).
Accordingly, the result reported in Fig. 2C demonstrates
that 32Dcl3 cells are unable to bind hIL-3.
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Figs. 3 and
4 show the results of experiments
performed to evaluate the in vivo angiogenic effect of hIL-3. Matrigel
containing 64 U/ml of heparin and 20 ng/ml hIL-3, 20 ng/ml hIL-3 plus
anti-hIL-3 Ab, 20 ng/ml hIL-3 plus WEB 2170, or sterile saline,
used as vehicle for hIL-3, was injected s.c. into mice. After 6 days,
mice were killed, and the Matrigel plugs were excised and processed for
histologic and morphometric analyses (Fig. 4). As shown in Fig. 4, hIL-3, in the presence of heparin, was able to induce neoangiogenesis,
while this angiogenic response was absent in Matrigel containing hIL-3
without heparin (data not shown). Several canalized vessels containing
erythrocytes and leukocytes were observed in the Matrigel plugs (Fig. 4, B–D). Some of the vessels formed microaneurysmatic
structures (Fig. 4, B and C); others were
surrounded by inflammatory cells (Fig. 4D). This effect was
absent in control mice treated with heparin plus saline (Fig. 4A). Moreover hIL-3-induced neoangiogenesis was
significantly reduced in mice injected with Matrigel containing hIL-3
plus anti-hIL-3 Ab (Fig. 4E) or hIL-3 plus WEB 2170
(Fig. 4F). Similar results were obtained when mouse IL-3
(mIL-3) or mIL-3 + anti-mIL-3 Ab was injected s.c. into mice (data
not shown). These in vivo results are consistent with the observation
that hIL-3 binds to murine endothelial cells and stimulates their in
vitro migration.
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The ability of hIL-3 to stimulate the synthesis of PAF was
evaluated. As shown in Fig. 5, HUVEC
synthesized PAF after stimulation with hIL-3. PAF synthesized after
hIL-3 stimulation remained all cell-associated, being undetectable in
the cell-free supernatant. Using radioactive acetate as substrate for
PAF synthesis, we found that PAF, detected after stimulation with
hIL-3, was newly synthesized. The TLC analysis of lipid fractions
extracted 4 h after addition of hIL-3 to HUVEC (Fig. 5)
preincubated with [3H]-acetate demonstrated the
presence of one main peak of radioactivity that comigrated with
synthetic [3H]-C16-PAF (data not shown). This
peak was absent in the lipid fractions extracted from unstimulated
HUVEC.
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To investigate the role of PAF in mediating hIL-3-induced
endothelial cell proliferation, the effects of hIL-3 in the presence or
in the absence of WEB 2170 was evaluated. As shown in Table II, in the absence of growth factor, no
significant increase in endothelial cell number was detected. By
contrast, as previously reported (8), hIL-3 was able to
support a 3-fold increase of the seeded cell number after 7 days of
culture. Moreover, the addition of WEB 2170 did not prevent
hIL-3-induced endothelial cell proliferation. A 5-fold increase of
endothelial cells was observed when bFGF, the reference growth factor,
was added to the cultures. Similar results were obtained when cell
proliferation was assessed by MTT colorimetric assay (Table II).
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Receptor activation leads to the recruitment of several
transducing molecules, including members of the STAT family
(14, 15, 16, 17). It has been shown that STAT5 proteins are
implicated in IL-3-induced cell proliferation (18). We
thus investigated the ability of hIL-3 to induce STAT5 protein
activation in endothelial cells. To this end, kinetic analysis of
STAT5A and STAT5B tyrosine phosphorylation in HUVEC stimulated with
hIL-3 was performed. As shown in Fig. 6,
A and B, both STAT5A and STAT5B became rapidly
tyrosine phosphorylated upon hIL-3 treatment; however, STAT5A tyrosine
phosphorylation was no more detectable after 15 min, while STAT5B
tyrosine phosphorylation was still detectable after 30 min of hIL-3
stimulation.
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demonstrate that both STAT5A
(A) and STAT5B (B) are physically associated with
the ßc subunit of the IL-3R and that this
interaction depends on ligand stimulation.
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Early studies on mammary gland tissue demonstrated that STAT5 was
able to recognize a specific DNA sequence located upstream of the ß
casein promoter, PIE (19). Therefore, to extend the
analysis of STAT5 protein activation in HUVEC, we evaluated, by gel
retardation assay, the formation of a DNA-protein complex with the PIE
sequence in nuclear extracts from untreated and hIL-3-treated cells.
The results shown in Fig. 8A
indicate that hIL-3 stimulation led to the formation of a PIE complex
in HUVEC. That the hIL-3-induced DNA-binding complex contained both
STAT5 proteins was evident from supershifted experiments in which Abs
to STAT5A and STAT5B were able to induce a mobility shift of the PIE
complex (Fig. 8B). As control for STAT5 protein activation,
hIL-3-stimulated M-07e cells were used (Fig. 8, A and
B).
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unstimulated HUVEC showed a peripheral cytoplasmic staining for STAT5A
(A) and STAT5B (C), by contrast a nuclear
staining in cells stimulated with hIL-3 indicates the ability of STAT5A
(B) and STAT5B (D) to migrate into the nucleus.
Controls, with preimmune rabbit serum or with an irrelevant isotypic
mouse IgG, used instead of the specific anti-STAT5 antisera, were
negative (data not shown).
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Our finding that hIL-3-induced angiogenesis is mediated by the
synthesis of PAF led us to investigate the role of this soluble
mediator on STAT5 protein activation in response to hIL-3. Unstimulated
and hIL-3-stimulated HUVEC, pretreated or not with WEB 2170, were
immunoprecipitated with specific antisera to STAT5A and STAT5B. That
WEB 2170 was unable to prevent IL-3-mediated STAT5A and STAT5B
activation was demonstrated by the anti-phosphotyrosine immunoblot
shown in Fig. 10, A and
B, respectively. In conclusion, these data indicate that the
effect of hIL-3 on STAT5 protein activation does not depend on the
synthesis of PAF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of hIL-3 are mediated by a heterodimeric receptor
consisting of a specific ligand-binding subunit denoted as
(9) and a transducing subunit, shared with the GM-CSF and
the IL-5 receptors, defined as ßc (10, 45). Cloning of the IL-3R in mice demonstrated the existence of
two different ß subunits (46), showing extensive
sequence homology to ßc of the human receptor.
By contrast, the ligand-binding () subunit of the murine receptor
(47) revealed a low homology with the human subunit
(30% identity) (9), in accordance with the low homology
between the murine and the human ligand (39). These
observations are consistent with the inability of hIL-3 to stimulate
proliferation and colony formation of murine hemopoietic cells (Ref.
39 , and our unpublished observations) and with the failure
of hIL-3 to bind to (Fig. 2
C) murine hemopoietic cells. On
the other hand, it has been also demonstrated that hIL-3, as the murine
IL-3, is a trophic factor for mouse central cholinergic neurons
(40), indicating that, in tissues different from bone
marrow, hIL-3 can bind to the mouse receptor. Indeed, the results
presented here demonstrate that hIL-3 can bind to murine endothelial
cells and is able to promote a motogenic response. Therefore, we
evaluated whether hIL-3 was also able to elicit an angiogenic response
in an experimental murine model. These in vivo studies demonstrated
that hIL-3-containing Matrigel injected s.c. in mice led to
neoangiogenesis and that, for a full expression of the in vivo
angiogenic properties, the presence of heparin was required (data not
shown). The hIL-3 dependency of the in vivo angiogenetic process was
confirmed by the inhibitory effect of anti-hIL-3 blocking Ab. Some
of the neo-formed vessels contained or were surrounded by inflammatory
cells consistently with the observation that hIL-3 triggers the
expression of adhesion molecules by endothelial cells (8, 43). Moreover, we found that a specific PAF receptor antagonist,
WEB 2170, was able to inhibit HUVEC migration in vitro and the
angiogenic process in vivo, suggesting that PAF may act as a mediator
of cell-to-cell communication of signaling induced, on endothelial
cells, by hIL-3. These results are reminiscent of those observed with
other polypeptide mediators that require the synthesis of PAF for the
full expression of their angiogenic properties (48, 49).
Indeed, we demonstrated that stimulation of HUVEC with hIL-3 leads to
the synthesis of PAF that peaks after 4 h of treatment, as
previously reported for IL-1 and TNF- (50). The role of
PAF in angiogenesis is mainly related to its motogenic activity on
endothelial cells, while the proliferative response required for new
vessel growth is sustained by the production of other
endothelial-derived heparin-dependent angiogenic factors
(48). In agreement with these observations, we found that
only the motogenic activity but not the proliferative effect of hIL-3
was prevented by a specific PAF receptor antagonist. Therefore, in this
context, hIL-3 may directly sustain the proliferative activity of
endothelial cells, whereas IL-3-mediated PAF release may account for
their migration. The observation that patients treated with hIL-3
showed an increase of endothelial cell number in bone marrow
microenviroment (51) supports the possibility that the
proliferative effect of hIL-3 on endothelial cells can also be
operative in vivo.
IL-3 binding to its receptor triggers a cascade of intracellular protein tyrosine phosphorylations, including members of the STAT family (14, 15, 16, 17). Although the functional role in specific cellular program of some STAT proteins has been demonstrated by targeting disruption of their genes, for the other members, considerable discussion centers on whether individual STAT proteins play a role in differentiation, proliferation, or both (15, 16, 17, 18). We found that in hIL-3-stimulated HUVEC both STAT5A and STAT5B are rapidly tyrosine phosphorylated and recruited by the ßc-transducing subunit of the hIL-3R before migrating into the nucleus to bind the target gene. Activation of STAT5 has been initially related to its ability to bind the ß casein promoter; however, more recent observations demonstrate that STAT5 can also interact with other DNA elements (18), suggesting that the role of STAT5 is not exclusively related to the lactating mammary genes. Indeed, STAT5 can interfere with the expression of the IL-3-mediated activation of early response genes (18). Similarly, a dominant negative STAT5 inhibits IL-3-stimulated cell proliferation (52). These observations, together with the finding that a constitutive activation of STAT5 correlated with the transition from IL-3-dependent to IL-3-independent growth of Ba/F3 cell line (53), suggest a role of STAT5 in regulating IL-3-induced mitogenic signals. It is then conceivable to assume that, also in endothelial cells, the activation of STAT5 proteins in response to hIL-3 may be associated with proliferative signals.
Inhibition of the transcriptional factor NF-
B has been shown to
block tubular morphogenesis of human microvascular endothelial cells
induced by oxidative stress, suggesting a role of this transcriptional
factor in regulating some events of neoangiogenesis (54).
Since PAF can activate NF-B (55, 56), it is possible
that NF-B may contribute to the proangiogenic effect of PAF. IL-3
does not directly activate NF-B (57); however, it may
activate this transcriptional factor indirectly via the synthesis
of PAF.
In conclusion, these results demonstrate that hIL-3 possesses angiogenic properties both in vitro and in vivo in a murine model of Matrigel implantation, suggesting a potential role of hIL-3 in sustaining new vessel formation in pathophysiological processes where T lymphocytes are involved. Moreover, the analysis of the mechanisms involved in hIL-3-induced angiogenic response indicates that, while endothelial cell migration depends on a PAF-mediated pathway, proliferation of endothelial cells is directly elicited by hIL-3.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Luigi Pegoraro, Dipartimento di Medicina Interna, Corso Dogliotti 14-10126 Torino, Italy. E-mail address:
3 Abbreviations used in this paper: PAF, platelet-activating factor; hIL-3, human hemopoietic growth factor IL-3; ßc, common ß; PIE, prolactin-inducible element; bFGF, basic fibroblast growth factor.
Received for publication March 26, 1999. Accepted for publication June 4, 1999.
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References |
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 J. H. Von der Thusen, J. Kuiper, T. J. C. Van Berkel, and E. A. L. Biessen Interleukins in Atherosclerosis: Molecular Pathways and Therapeutic Potential Pharmacol. Rev., March 1, 2003; 55(1): 133 - 166. [Abstract] [Full Text] [PDF]
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 I. HARTLAPP, R. ABE, R. W. SAEED, T. PENG, W. VOELTER, R. BUCALA, and C. N. METZ Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote angiogenesis in vivo FASEB J, October 1, 2001; 15(12): 2215 - 2224. [Abstract] [Full Text] [PDF]
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 M. F. Brizzi, L. Formato, P. Dentelli, A. Rosso, M. Pavan, G. Garbarino, M. Pegoraro, G. Camussi, and L. Pegoraro Interleukin-3 Stimulates Migration and Proliferation of Vascular Smooth Muscle Cells : A Potential Role in Atherogenesis Circulation, January 30, 2001; 103(4): 549 - 554. [Abstract] [Full Text] [PDF]
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 G. Montrucchio, G. Alloatti, and G. Camussi Role of Platelet-Activating Factor in Cardiovascular Pathophysiology Physiol Rev, October 1, 2000; 80(4): 1669 - 1699. [Abstract] [Full Text] [PDF]
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)
or vehicle alone (
). PAF was extracted at different time intervals,
purified by TLC, and tested by bioassay, as described in
Materials and Methods. Data are expressed as the
mean ± SD of three individual experiments.
