Introduction
Angiogenesis is a complex multistep phenomenon consisting of the sprouting and the growth of new capillary blood vessels starting from the pre-existing ones. It requires the cooperation of several cell types such as endothelial cells (ECs), vascular smooth muscle cells (VSMCs), macrophages, which should be activated, proliferate and migrate to invade the extracellular matrix and cause vascular remodeling [
1,
2]. The angiogenic process is finely tuned by a precise balance of growth and inhibitory factors and in mammalians it is normally dormant except for some physiological conditions, such as wound healing and ovulation. When this balance is altered, excessive or defective angiogenesis occur and the process becomes pathological. Excessive angiogenesis gives also rise to different dysfunctions, including cancer, eye diseases, rheumatoid arthritis, atherosclerosis, diabetic nephropathy, inflammatory bowel disease, psoriasis, endometriosis, vasculitis, and vascular malformations [
3]. Therefore the discovery of angiogenesis inhibitors would contribute to the development of therapeutic treatments for these diseases.
The integrins are cell adhesion receptors that mediate cell-cell and cell-matrix interactions and coordinate signaling allowing a close regulation of physiological phenomena including cellular migration, proliferation and differentiation. In particular, the α
V integrins, combined with distinct β subunits, participate in the angiogenic process. An extensively studied member of this receptor class is integrin α
Vβ
3, that is strongly overexpressed in activated EC, melanoma, glioblastoma and prostate cancers and in granulation tissue, whereas is not detectable in quiescent blood vessels or in the dermis and epithelium of normal skin [
4‐
6]. This integrin participates in the activation of vascular endothelial growth factor receptor-2 (VEGFR-2), providing a survival signal to the proliferating vascular cells during new vessel growth [
7,
8] and also seems to be essential in the step of vacuolation and lumen formation [
9]. It has been also reported that α
Vβ
3 is under the tight control of VEGF: this integrin is not expressed in quiescent vessels [
10], but VEGF induces α
Vβ
3 expression
in vitro and, interestingly, the VEGF and α
Vβ
3 integrin expression are highly correlated
in vivo [
11,
12]. Therefore, α
Vβ
3 should be considered a tumor and activated endothelium marker.
α
Vβ
3 is able of recognizing many proteins of the extracellular matrix, bearing an exposed Arg-Gly-Asp (RGD) tripeptide [
5,
13,
14]. Even if different integrins recognize different proteins containing the RGD triad, many studies have demonstrated that the aminoacids flanking the RGD sequence of high-affinity ligands appear to be critical in modulating their specificity of interaction with integrin complexes [
15,
16].
Several molecules including peptides containing RGD motif [
11] have been recently developed as inhibitors of α
Vβ
3 integrin, in experiments concerning tumor angiogenesis, showing a reduction of functional vessel density associated with retardation of tumor growth and metastasis formation [
6,
17]. So far, the pentapeptide c(RGDf[NMe]V), also known as cilengitide (
EMD 121974), is the most active α
vβ
3/α
vβ
5 antagonist reported in literature [
18,
19] and is in phase III clinical trials as antiangiogenic drug for glioblastoma therapy [
15]. The development of more selective antiangiogenic molecule would help to minimize the side-effects and increase the therapeutic effectiveness.
We have recently designed and synthesized a novel and selective peptide antagonist, referred to as RGDechiHCit, to visualize α
Vβ
3 receptor on tumour cells [
20]. It is a chimeric peptide containing a cyclic RGD motif and two echistatin C-terminal moieties covalently linked by spacer sequence. Cell adhesion assays have shown that RGDechiHCit selectively binds α
Vβ
3 integrin and does not cross-react with α
Vβ
5 and α
IIbβ
3 integrins [
20]. Furthermore, PET and SPECT imaging studies have confirmed that the peptide localizes on α
Vβ
3 expressing tumor cells in xenograft animal model [
21]. Since α
Vβ
3 is also a marker of activated endothelium, the main purpose of this study was to evaluate
in vitro and
in vivo effects of RGDechiHCit on neovascularization. Thus, we first assessed the
in vitro peptide properties on bovine aortic ECs, and then
in vivo, in Wistar Kyoto (WKY) rats and c57BL/6 mice, the ability of this cyclic peptide to inhibit angiogenesis.
Discussion
In the present study, we evaluated the anti-angiogenic properties of RGDechiHCit peptide
in vitro on EC and VSMC cells and
in vivo on animal models of rats and mice. The data here reported recapitulate the well-known antiangiogenic properties of c(RGDf[NMe]V), that was used as control. We previously described the design and synthesis of RGDechiHCit, a novel and selective ligand for α
Vβ
3 integrin, containing a cyclic RGD motif and two echistatin
C-terminal moieties [
20].
In vitro studies showed that this molecule is able to selectively bind α
Vβ
3 integrin and not to cross-react with other type of integrins. Furthermore, PET and SPECT imaging studies have confirmed that the peptide localizes on α
Vβ
3 expressing tumor cells in xenograft animal model [
21]. Given the presence in the molecule of the RGD sequence it was obvious to speculate that RGDechiHCit acted as an antagonist. Our report is the first evidence that our peptide acts as antagonist for α
Vβ
3 integrin. Its ability to inhibit hFN-induced cell proliferation is comparable to that of c(RGDf[NMe]V), although the half-life is quite reduced.
A major evidence that is brought up by our results is the peculiar selectivity of RGDechiHCit towards EC, as compared to c(RGDf[NMe]V). Indeed, RGDechiHCit fails to inhibit VSMC proliferation
in vitro, opposite to c(RGDf[NMe]V). We believe that this feature is due to the selectivity of such a novel compound toward α
Vβ
3. Indeed, VSMCs express α
Vβ
3 only during embryogenesis [
31], but express other integrins which may be blocked by c(RGDf[NMe]V). On the contrary, α
Vβ
3 is expressed by ECs [
8], thus conferring RGDechiHCit selectivity toward this cell type. This issue is relevant cause the effect
in vivo is similar between the two antagonists on wound healing and Matrigel plugs invasion. Indeed, our data suggest that inhibition of the endothelial integrin system is sufficient to inhibit angiogenesis. It is possible to speculate that the higher specificity of RGDechiHCit for the endothelium would result in a lower occurrence of side effects than the use of less selective inhibitors. This is only an indirect evidence, that needs further investigation in more specific experimental setups. Indeed, of the wide spectrum of integrins that are expressed on the surface of ECs, α
Vβ
3 receptor has been identified as having an especially interesting expression pattern among vascular cells during angiogenesis, vascular remodeling, tumor progression and metastasis [
6,
32,
33]. What is more, two pathways of angiogenesis have been recently identified based on the related but distinct integrins α
Vβ
3 and α
Vβ
5 [
4]. In particular, α
Vβ
3 integrin activates VEGF receptors and inhibition of β
3 subunit has been shown to reduce phosphorylation of VEGF receptors [
7], thereby limiting the biological effects of VEGF [
1]. Further, Mahabeleshwar and coworkers have shown the intimate interaction occurring between α
Vβ
3 integrin and the VEGFR-2 in primary human EC [
12]. The relevance of this molecule to angiogenesis and its potential as a therapeutic target has, therefore, been well established [
34,
35] and in this report we show that its activity is highly critical for both hFN or VEGF-stimulated ECs proliferation.
Our results concerning RGDechiHCit in angiogenic processes are of immediate translational importance, because deregulation of angiogenesis is involved in several clinical conditions including cancer, ischemic, and inflammatory diseases (atherosclerosis, rheumatoid arthritis, or age-related macular degeneration) [
34‐
36]. Therefore, the research for drugs able to modulate angiogenesis constitutes a crucial investigation field. Since RGDechiHCit is rapidly removed in serum it is possible to increase its effect by engineering the molecule to elongate its lifespan. In the present paper we circumvented this issue by increasing the times of application of the drug both
in vitro and
in vivo, or by reducing the times of observation. This issue can be solved by the use of a more stable aromatic pharmacophore that recapitulates the binding properties of RGDechiHCit. Clearly, further investigations are also needed to fully understand the basic cell biological mechanisms underlying growth factor receptors and integrin function during angiogenesis. The knowledge of molecular basis of this complex mechanism remains a challenge of fascinating interest, with clinical implications for treatment of a large number of pathophysiological conditions including but not limited to solid tumors [
17,
37], diabetic retinopathy [
38,
39] and inflammatory disease [
36].
Competing interests
We have no financial or personal relationships with other people or organizations that would bias our work. No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of our article.
Authors' contributions
GS and GI designed research; GS, MFB, MDS, CDG, AA, and DS carried out the experiments; GS and GI performed the statistical analysis; GS, GI and LZ drafted the manuscript; GS, MS, ADG, BT, CP and GI supervised the project; GS and MFB equally contributed to this work. All authors read and approved the final manuscript.