Introduction
Therapeutic vascular growth is a novel rising area for the treatment of ischemic vascular diseases. Limited options for treatment of chronic ischemic diseases, in particular in patients with severe atherosclerosis, have induced to study new therapeutic approaches based on the possibility to increase the development of collateral circulation [
1]. This complex process involves both angiogenesis (creation of new capillaries) and arteriogenesis (enlargement and remodeling of pre-existing collaterals) [
2]. In detail, the term angiogenesis refers to the sprouting, enlargement, or intussusceptions of new endothelialized channels and is tightly associated to endothelial cells proliferation and migration in response to angiogenic stimuli, in particular hypoxia. Arteriogenesis is, instead, a result of growth and positive remodeling of pre-existing vessels, forming larger conduits and collateral bridges between arterial networks via recruitment of smooth muscle cells. Unlike angiogenesis, this process is linked to shear stress and local activation of endothelium rather than hypoxia [
3]. Nevertheless, these two mechanisms interplay during conditions of chronic ischemia and can be modulated by several growth factors, transcription factors and cytokines [
3,
4].
In particular, the main regulator of neovascularization in adult life is the system of vascular endothelial growth factor (VEGF), that is expressed as several spliced variants. Among its several isoforms, VEGF
165 is the one that until now has shown the ability to regulate mechanisms of neovascularization both
in vitro and
in vivo. The two main VEGF receptors are VEGFR-1 or fms-like tyrosine kinase 1 (Flt-1) and VEGFR-2 or fetal liver kinase 1 (Flk-1) also known as kinase-insert domain-containing receptor (KDR) [
2].
In animal models of chronic ischemia, manoeuvres that increase VEGF levels by intramuscular injection or vascular infusion of adenoviral vectors encoding for VEGF [
5,
6], or indirectly, for example by physical training or β
2 adrenergic receptor overexpression in ischemic hindlimb (HL), have shown to improve collateral flow [
3,
5‐
7]. In spite of all, clinical trials using gene or protein therapy with VEGF isoforms for treatment of myocardial or peripheral ischemia have been somewhat disappointing indicating the needs to develop new approaches in this field [
1,
8].
We recently demonstrated that a
de novo synthesized VEGF mimetic, named QK, shares the same biological properties of VEGF and shows the ability to induce capillary formation and organization
in vitro [
9], and showed to be active in gastric ulcer healing in rodents when administered either orally or systemically [
10]. This mimetic is a 15 amino acid peptide which adopts a very stable helical conformation in aqueous solution [
11] that resembles the 17–25 α-helical region of VEGF
165, and binds both VEGFR-1 and 2.
The main purpose of this study is to evaluate
in vivo the effects of this
de novo engineered VEGF mimicking peptide on neovascularization, in normotensive Wistar Kyoto (WKY) rats. Therefore, we first assessed the properties of QK performing
ex vivo experiments of vascular reactivity in WKY common carotid rings [
12], and then we evaluated
in vivo the role of this small peptide studying the angiogenic models of ischemic HL, wound healing and Matrigel plugs.
Discussion
In the present study, we examinated the
in vivo effects of a VEGF
165 mimetic, named QK, modeled on the region of the VEGF protein responsible for binding to and activating the VEGFRs that are known to trigger angiogenesis. We previously showed that QK can bind to the VEGFRs, initiate VEGF-induced signaling cascades and stimulate angiogenesis
in vitro [
9]. This is the first report to show that this peptide is able to recapitulate the
in vivo responses of VEGF.
Angiogenesis is known to be a process of new blood vessel formation from a pre-existing endothelial structure. It is tuned by proangiogenic and antiangiogenic factors, and the shift from this equilibrium may lead to pathological angiogenesis [
18,
19]. Indeed, deregulation of angiogenesis is involved in several conditions including cancer, ischemic, and inflammatory diseases (atherosclerosis, rheumatoid arthritis, or age-related macular degeneration). Therefore, the research for drugs able to regulate angiogenesis constitutes a pivotal research field. In particular, occlusive vascular disease remains an important cause for death and morbidity in industrialized society [
1,
20], despite efforts to design new and efficient treatment strategies [
19,
21].
Unfortunately, numerous reports indicate that in laboratory animals over-expression of VEGF may lead to metabolic dysfunction, formation of leaky vessels and transient edema [
1,
22]. Indeed, VEGF actions include the induction of endothelial cells proliferation and migration; it is also known as a vascular permeability factor, based on its ability to induce vascular leakage and vasodilatation in a dose dependent fashion as a result of endothelial cell-derived nitric oxide [
12,
23].
In humans, various clinical trials were designed to verify new vessel growth by exogenous administration of proangiogenic factors in patients with refractory ischemic symptoms. Albeit initial small open-labeled trials yielded promising results, subsequent larger double-blind randomized placebo-controlled clinical trials have failed to show much clinical benefit [
19,
24,
25]. These largely disappointing results may in part be explained by suboptimal delivery of genetic material to target cells or tissue. Moreover, although adenoviral vectors provide high levels of gene transfer and expression, there are well known virus-related adverse effects, such as the induction of immune and inflammatory response [
6,
21,
26]. Recently, several side effects have been reported for VEGF administration in human subjects [
1,
8,
25] such as increase in atherosclerotic plaques, lymphatic edema or uncontrolled neoangiogenesis leading to the development of functionally abnormal blood vessels, so to preclude its use in a large share of ischemic population [
21,
27].
A hopeful alternative could be to use angiogenic stimulators of smaller size, such as peptides, with a well-characterized biologic mechanism of action. Indeed, recent reports revealed a specific antagonistic relationship between VEGF and other vascular growth factors, such as the placental growth factor (PlGF), the basic fibroblast growth factor (bFGF) and the platelet-derived growth factor (PDGF), with a dichotomous role for VEGF and VEGFRs [
28‐
30]. So, the function of VEGF is far more intricate: it can also negatively regulate angiogenesis and tumorigenesis, by impeding the function of the PDGF receptor on pericytes, leading to a loss of pericyte coverage of blood vessels [
31]. Moreover, several studies demonstrated a more efficacious action obtained with a specific stimulation of VEGFRs [
32,
33] if compared to VEGF overexpression [
22,
34]. These findings suggest that the multifaceted array of the biological responses linked to VEGF may be ascribable to its proneness to dimerize or interact with other molecules [
29]. Thus, because of lower molecular and biological complexity, peptides that ensure only the needed interaction with specific receptors could be candidate lead compounds for a safer proangiogenic drug, also to avoid adverse effects.
Perspectives
We show that the VEGF mimetic QK is able to increase neoangiogenesis and collateral flow in WKY rats. Our findings evidence the proangiogenic properties of this small peptide, suggesting that also in vivo QK resembles the full VEGF protein. Thus, a single peptide, that would not be expected to dimerize, is still able to induce VEGF specific angiogenic responses. Clearly, further studies are needed to fully understand this mechanism, that appears of intriguing interest. Anyway, these data open to new fields of investigation on the mechanisms of activation of VEGFRs, also to clarify complex angiogenesis pathways, with strong clinical implications for treatment of pathophysiological conditions such as chronic ischemia.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
GS, GI, MC, LDDA, CP and BT designed research, GS, MC, GP, AC, GG, BZ, GGA, VC, and FP, carried out the experiments; GS and GI performed the statistical analysis; GS, GI and BT drafted the manuscript. All authors read and approved the final manuscript.