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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 24, 2004 as doi:10.1096/fj.04-2114fje. |
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Pharmacology Section, Department of Molecular Biology, University of Siena, Siena, Italy; and
* Department of Biomedical Sciences and Biotechnologies, School of Medicine, University of Brescia, Brescia, Italy
2Correspondence: Department of Molecular Biology, University of Siena, Via A. Moro 2, 3100 Siena, Italy. E-mail: ziche{at}unisi.it
SPECIFIC AIMS
Amyloid ß peptides (Aß peptides), widely recognized as one of the causative factors of Alzheimer’s disease, are produced by a variety of cells and are naturally present in low nanomolar quantities in the cerebrospinal fluid and blood of healthy individuals. Although a plethora of studies have investigated Aß peptides in relation to Alzheimer’s disease, the physiological role of Aß peptides remains poorly defined.
The aim of the study was to investigate the effects of Aß peptides (Aß1-40 and Aß1-42) and of the soluble synthetic fragment Aß25-35 on endothelial cell functions required for angiogenesis. This study highlights an important and specific effect of low concentrations of the Aß peptides on the process leading to neovascularization. Our results indicate a selective synergism between the Aß peptides and fibroblast growth factor-2 (FGF-2) in promoting angiogenesis in vivo and reveal an unexpected protective role of Aß peptides in sustaining microvascular growth.
PRINCIPAL FINDINGS
1. Aß peptides promote endothelial cell growth, migration, and microtubule network formation
We assessed the effect of Aß peptides on endothelial proliferation and migration and on the formation of new capillaries. Exposure of quiescent endothelial cells (CVEC) to Aß1-40 or its synthetic partial sequence Aß25-35 stimulated proliferation at nanomolar concentrations whereas micromolar concentrations inhibited growth (Fig. 1
A). Aß1-40 or Aß25-35 enhanced cell proliferation in a manner comparable to that of FGF-2 or vascular endothelial growth factor (VEGF).
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In cell migration experiments, all Aß peptides more than doubled the number of migrating cells compared with control (0.1% BCS), their effect being greater than that of VEGF or FGF-2 (Fig. 1B
). Consistently, Aß1-40, Aß1-42, or Aß25-35 stimulated the formation of capillary-like structures in explants from mouse aorta to an extent comparable to that of 10% FCS (Fig. 1C
). The effect induced by Aß1-42 is exemplified in Fig. 1D
. Reverse sequences Aß40-1 or Aß35-25 had no effect on proliferation, migration or vessel formation (Fig. 1A-C
).
To elucidate the effect of Aß peptides on endothelial cell growth and migration, we assessed their activity on ERK1/2 phosphorylation and cdk2 expression, matrix metalloproteinase (MMP) and endothelial nitric oxide synthase (eNOS) activities. Aß1-40 or Aß25-35 (50 nM) doubled ERK1/2 phosphorylation and cdk2 expression and promoted matrix metalloproteinase (MMP-2 active form) release. Together, these data indicate that Aß peptides induce endothelial cells to acquire an angiogenic phenotype by activating ERK1/2 and MMP-2 release.
2. Aß peptides induce endothelial cell growth via FGF-2 signaling pathway
Experiments measuring endothelial cell proliferation or pseudocapillary formation evidenced that Aß peptides angiogenic activity strictly depends on FGF-2 availability. In fact, prior exposure to an antibody against FGF-2 abolished cell growth and prevented pseudocapillary formation. The antibody also decreased ERK phosphorylation, clearly demonstrating the absolute requirement of FGF-2 for the full expression of Aß activity.
At a molecular level, Aß peptides specifically promoted phosphorylation of c-Raf on serine 338, an upstream signal specific for FGF-2, and up-regulated by 6-fold FGF-2 mRNA levels in endothelial cell. This marked up-regulation was totally abolished by the anti-FGF-2 neutralizing antibody. Thus, all this evidence clearly demonstrates a close linkage between Aß peptide activity and FGF-2 signaling and transcription.
3. Aß peptides synergize with FGF-2 in promoting endothelial cell growth and migration in vitro
To elucidate the interplay between Aß peptide activities and FGF-2, we investigated whether Aß peptides cooperate with FGF-2 in inducing endothelial cell proliferation and migration. Low concentrations of Aß1-40, or Aß25-35 (both at 0.5 nM) did not alter cell proliferation, whereas when the peptides were combined with a marginally effective FGF-2 concentration (3 ng/mL), a marked increment in cell count over basal occurred (Aß25-35 5±3, FGF-2 19±4, Aß25-35+FGF-2 73±5; P<0.001). In contrast, combined treatment with VEGF (5 ng/mL) and Aß peptides did not produce an increase of cell number. Migration experiments substantiated the synergistic effect between Aß and FGF-2. At the molecular level, a dramatic increase in phosphorylation of ERK1/2 was consistently observed when ineffective concentrations of both Aß and FGF-2 were combined, an effect not shared by VEGF.
4. Aß peptides potentiate FGF-2-induced angiogenesis in vivo
We finally designed experiments in vivo in the rabbit cornea to analyze the interaction between Aß peptides and FGF-2 or VEGF. We observed a striking synergistic response when FGF-2 and Aß1-40 were coreleased from adjacent pellets containing 50 ng of each peptide in a single rabbit eye (Fig. 2
A, D) When released separately, neither molecule elicited angiogenesis (Fig. 2A
). Conversely, no potentiation occurred when Aß1-40 and VEGF were simultaneously delivered to the corneal stroma at threshold doses, which per se elicited only a negligible response (Fig. 2C, F
). These findings reinforce the specificity of Aß/FGF-2 interaction. The failure of the reverse peptide Aß40-1 to elicit an angiogenic response and to synergize with FGF-2 indicates the molecular specificity of the interaction (Fig. 2B, E
).
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CONCLUSIONS
Aß peptides form insoluble aggregates responsible for plaque formation in Alzheimer’s disease. Accumulation of misfolded amyloid fibrils is generally believed to be a key pathogenic event in several brain disorders. A wealth of studies have reported that high concentration of Aß peptides negatively affect the functioning of cells, including endothelial cells, producing toxic injuries leading to cell death. Because Aß peptides are ubiquitously produced, and found at low concentrations in the blood of healthy subjects, we speculated that they may have a physiological function on the vasculature.
We present evidence that amyloid peptides strongly activate the vascular endothelium at the cellular, biochemical, and in vivo integrated level, leading to new vessel formation. Our study demonstrates that nanomolar concentrations of Aß peptides induce an angiogenic phenotype in cultured endothelial cells, exemplified by enhanced proliferation, induction of cell cycle markers, increased migration, and activation of MMP-2 activity (Fig. 3
). Aß not only mimicked the effect of the growth factors FGF-2 but also induced it, required it, and synergized with it.
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In fact, endothelial cell exposure to combined threshold concentrations of Aß peptides and FGF-2 produced proliferative and migratory effects that greatly exceeded those obtained by suboptimal concentrations of the individual molecules. The interaction was highly specific as neither VEGF nor the scramble sequence of the peptides produced synergism. The activity of A
ß peptides on microvascular endothelium appeared to be critically dependent on the presence of either endogenous or exogenously administered FGF-2 as its blockade, through neutralizing antibodies, completely abolished the stimulated formation of pseudocapillaries in organ explants and endothelial cell functions. Pronounced synergism between Aß peptides and FGF-2 was evident in vivo using the rabbit cornea assay, resulting in robust growth of new vessels.
At the biochemical level Aß peptides induced activation of signals typically used by FGF-2 to promote the endothelial angiogenic phenotype. In fact, the observed activation of the Raf pathway selectively involved the Raf pSer338/339 species, leaving the NOS pathway unaffected. Collectively, these findings beside indicating the preference of Aß peptides for the FGF-2 signaling pathway, provide a clear rationale for their failure to interact and synergize with VEGF. In all, these data unravel a novel functional interplay between Aß peptides and FGF-2 signaling pathways.
The Aß peptides emerge as physiological molecules capable of promoting vigorous angiogenesis in microvascular endothelium, through interaction with endogenous FGF-2 and induction of its synthesis. Studies showing that FGF-2 increases endothelial survival contributed to the notion of FGF-2 as a tonically produced factor that protects the endothelium through activation of Raf kinases. The finding that Aß peptides participate in FGF-2 activities indicates that this molecule is involved along with others in the inherent endothelial cell survival program and delineate a role for circulating Aß peptides as important primers of the FGF-2 cycle. The relevance of these observations to microangiopathy and Alzheimer’s disease, both associated with Aß peptides dysregulation, remains to be explored. Nevertheless, the recognition that the Aß peptide/FGF-2 interaction has an important role in promoting angiogenesis and in maintaining microvascular integrity, might have implications for understanding the initial stages of Alzheimer’s disease and for the design of therapies targeting ß amyloid.
FOOTNOTES
1 These authors contributed equally to this work. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2114fje;
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  S. Donnini, R. Solito, A. Giachetti, H. J. Granger, M. Ziche, and L. Morbidelli Fibroblast Growth Factor-2 Mediates Angiotensin-Converting Enzyme Inhibitor-Induced Angiogenesis in Coronary Endothelium J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 515 - 522. [Abstract] [Full Text] [PDF]
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P < 0.001 vs. FGF-2 alone, &P < 0.001 vs. Aß1-40 alone. No statistical significance found in results of panels B, C.