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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2001 as doi:10.1096/fj.01-0633fje. |
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Institute for Cancer Research and Treatment and Department of Genetics, Biology and Biochemistry, School of Medicine, University of Torino, Torino, Italy; and
* Department of Biomedical Sciences and Human Oncology, School of Medicine, and
Department of Human Anatomy and Histology, University of Bari, Bari, Italy
2Correspondence: I.R.C.C., s.p. 142, Km 3.95, 10060 Candiolo, Italy. E-mail: fbussolino{at}ircc.unito.it
SPECIFIC AIMS
Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) induces the expression of a functional program in cultured endothelial cells (ECs) related to angiogenesis and to the their survival in the bone marrow microenvironment. Recently we have reported that human ECs express the specific GM-CSF receptor that signals through the recruitment and the activation of Janus kinase (JAK)2. The aim of this work was to demonstrate that the involvement of this cytosolic kinase and its downstream effector signal transducers and activators of transcription (STATs) is also operative during in vivo angiogenesis in chick chorioallantoic membrane (CAM).
PRINCIPAL FINDINGS
1. GM-CSF induces an angiogenic response in the CAM assay
On incubation day 12, the gelatin sponges treated with GM-CSF at 200–400 ng/sponge or with VEGF-A165 at 500 ng/sponge were surrounded by allantoic vessels radiating toward the implant in a ‘spoked-wheel’ pattern (Fig. 1
A, B). In the specimens treated with PBS, no vascular reaction was detectable around the sponges (Fig. 1C
). Microscopically, a highly vascularized tissue was observed among the trabeculae of the sponges treated with GM-CSF or VEGF-A165 consisting of newly formed blood vessels, mainly 3–10 µm capillaries growing perpendicular to the plane of the CAM within an abundant network of collagen fibers (Fig. 1D
, 1E
). The vessels were absent among trabeculae of implants treated with PBS (Fig. 1F
). The number of infiltrating leukocytes was negligible in control and stimulated CAM (not shown).
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In agreement with the macroscopic and microscopic observations, morphometry demonstrated that microvessel density in the sponges treated with 400 ng/implant of GM-CSF was comparable to that of the sponges treated with 500 ng/implant of VEGF165, a well-known angiogenic inducer (% microvessel density: control: 0; GM-CSF: 25±3; VEGF-A165:27±4).
2. GM-CSF induces endothelial cell sprouting from chicken aorta
To gain further direct insight into cellular targets of GM-CSF in the avian model, we evaluated the effect of GM-CSF on endothelial sprouting from adult chicken aorta. This is a well-accepted model to study the effects of angiogenic inducers since it allows direct monitoring of microvascular sprouting. After embedding chicken aorta in growth factor-reduced Matrigel, branching microvessels developed at the periphery of aortic rings treated with M199 medium 20% FCS, used as positive control, as well as with serum-free medium containing 50 ng/ml GM-CSF. In preliminary experiments, this concentration has been demonstrated to be the lowest able to give the maximal effect. In contrast, no vessel formation was evident in aortic rings cultured with serum-free medium alone. Quantitative analysis of microvessel length at different times revealed a clear-cut induction of vessel growth after stimulation of aortic rings with GM-CSF [length of outgrowing microvessels at 4th day (mean±SD): GM-CSF (50 ng/ml): 631 ± 89 µm; control: 32 ± 12].
3. GM-CSF activates tyrosine phosphorylation of JAK-2 in CAM
We have previously demonstrated that in vitro GM-CSF rapidly activates the catalytic activity of JAK-2 in human ECs. To examine the role of this tyrosine kinase during angiogenesis, we measured JAK-2 activity in 8-day-old chick CAMs stimulated with GM-CSF. Detergent-solubilized proteins of these CAMs were immunoprecipitated with anti-JAK-2 antibody, separated by SDS-PAGE, blotted, and analyzed by anti-phosphotyrosine antibody. GM-CSF (20 ng/ml) was able to induce the tyrosine phosphorylation of a 130 kDa protein specifically recognized by anti-JAK-2 antibody. Time course experiments demonstrated that JAK-2 tyrosine phosphorylation elicited by GM-CSF was evident after 10 min, reaching a maximum after 15 min (Fig. 2
A). This effect was clearly evident at subnanomolar concentrations, following a bell-shaped dose-dependent curve. Tyrosine phosphorylation of JAK-2 was detected in CAMs challenged with 5 ng/ml GM-CSF and dramatically increased with 50 ng/ml. At 200 ng/ml GM-CSF, the effect on JAK-2 was similar to the basal values. Stimulation of CAMs with heat-inactivated GM-CSF (50 ng/ml) was ineffective. No precipitation of JAK-2 was detected with nonimmune rabbit antiserum (not shown).
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4. GM-CSF activates tyrosine phosphorylation of STAT-3 in CAM
Since STAT-3 and STAT-5 are cytosolic effectors of GM-CSF and are phosphorylated by JAK-2, we examined whether their tyrosine phosphorylation occurred in 8-day-old CAM. The phosphorylation of STAT-3 was low in unstimulated tissues; after GM-CSF addition to CAMs, it appeared after 10 min and lasted <30 min (Fig. 2B
). The dose dependence STAT-3 phosphorylation paralleled that described for JAK-2. In fact, the greatest phosphorylation was observed with 20–50 ng/ml GM-CSF and was negligible at 200 ng/ml. STAT-5 phosphorylation in tyrosine residues was present in unstimulated CAMs and was not modified by GM-CSF stimulation.
CONCLUSIONS
GM-CSF is a cytokine that participates in myeloid differentiation and regulation of leukocyte functions. In addition, it plays key roles in activating genic programs in ECs, particularly those of bone marrow.
GM-CSF heterodimeric receptor is composed by two transmembrane glycoprotidic subunits, termed 
and ß. The 60–90 kDa chain specifically binds the ligand with low affinity; after binding the cytokine, it dimerizes with the 120–140 kDa ß chain. Although GM-CSFR lacks intrinsic catalytic activities, ligand-induced receptor dimerization is believed to mediate activation of receptor-associated JAK-2. Besides the ß chain itself, the most important substrates of JAK-2 are latent transcription factors (STATs). GM-CSF induces the JAK-2-dependent tyrosine phosphorylation of STAT-1, -3, and -5. STATs are normally located in the cytosol and bind the activated GM-CSFR, where they are phosphorylated by receptor-associated JAK kinases. Upon phosphorylation, STATs dissociate from the receptor and form homodimers and heterodimers that translocate to the nucleus, where they modulate target gene expression.
We have previously demonstrated that in human ECs, the amount of JAK2 physically associated with GM-CSFR ß chain is increased after GM-CSF stimulation and that picomolar concentrations of GM-CSF trigger a rapid activation of catalytic activity. Because GM-CSF activates EC migration and proliferation, we show evidence that in vivo activation of the JAK-2/STAT-3 pathway occurs during angiogenesis triggered by GM-CSF. This is based on the following observations: 1) GM-CSF triggers angiogenesis in chick embryo CAMs and induces vessel sprouting from chicken aorta; 2) activation of JAK2, evaluated by the autophosphorylation in tyrosine residues, is significantly enhanced in CAM after few minutes by GM-CSF stimulation used in the same range of concentration (subnanomolar amounts) able to activate angiogenesis and vessel sprouting; 3) STAT-3, which is a cytosolic effector of GM-CSF-dependent cell activation, is also tyrosine phosphorylated in vivo, with a delayed time course but for a longer period; 4) AG-490, an inhibitor of JAK-2, abrogates the angiogenesis induced by GM-CSF but is ineffective when VEGF-A165 is used as a stimulus.
These data represent the first demonstration of a role of JAK-2/STAT-3 pathway in angiogenesis (Fig. 3
).It is intriguing that STAT-3 has been implicated in the anti-apoptotic phenomena in ECs by up-regulating the expression of survivin. VEGF-A and hepatocyte growth factor, two well-known activators of angiogenesis that induce proliferation, migration, and survival of ECs, cause the phosphorylation of STAT-3. Survival factors are crucial in the stabilization of nascent vessels and prevent their regression, which may occur in the early steps of angiogenesis when pericytes are not yet recruited and the extracellular matrix is not completely remodeled.
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The data presented here do not give information on the cellular target of GM-CSF in CAM. The angiogenic effect may be related to a stimulation of preexisting CAM ECs or to a stimulation of hematopoietic cells carrying GM-CSFR (i.e., monocytes/macrophages) that release angiogenic inducers. However, the absence of detectable infiltrating leukocytes in GM-CSF-stimulated CAM may indicate that ECs are the most important target in our experimental model. It will be interesting to see which molecular events related to the induction of the angiogenic program in ECs are regulated by this signaling pathway.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0633fje; to cite this article, use FASEB J. (December 14, 2001) 10.1096/fj.01-0633fje 
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