The vascular endothelial growth factor (VEGF) system acts by specific VEGF receptors belonging to the superfamily of tyrosine-kinase receptors and represents a key regulator of angiogenesis processes. At the level of the vascular wall, the VEGF is produced by parenchymal cells under control of several triggers, including hypoxia and platelet-derived growth factor. The angiogenic activity of the system is directed to local endothelial cells by promoting their migration and proliferation . Beyond control of endothelial cell proliferation, VEGF also influences endothelial cell physiology, increasing the expression and activity of endothelial nitric oxide (NO) synthase, ultimately leading to the generation and release of NO and consequent vasodilatation . Among its wide spectrum of activities, NO counterbalances the vasoconstricting activity of endothelin-1 (ET-1)  and stimulates the production of tissue plasminogen activator (t-PA) and its main inhibitor, the plasminogen activator inhibitor type 1. In healthy vessels, a rapid release of t-PA is a counterregulatory mechanism to clear intravascular fibrin, thereby preventing thrombus extension . Therefore, in physiological conditions, the VEGF system contributes to the control of vascular homeostasis by modulating the NO/ET-1 axis and acts as a regulator of fibrinolysis.
In the last decades, a large body of literature documented that the VEGF signalling may also dysregulate angiogenesis, which represents a fundamental contributor to growth of cancer and neoplastic proliferation and invasion. This evidence has led to the development of new inhibitors of angiogenetic mechanisms targeting the VEGF signalling . VEGF inhibitors represent a novel and valuable strategy in the treatment of several malignancies, in which it has contributed to their improved survival. Bevacizumab was the first anti-VEGF mAb available in clinical practice and is now widely used in the treatment of several solid cancers. However, since their initial use, these drugs have shown a clinically relevant toxicity, especially hypertension. Several potential mechanisms might account for the increase in blood pressure (BP) observed in patients treated with bevacizumab. One of them is a VEGF inhibitor-induced upregulation of the ET-1 vasoconstrictor function while another is the abrogation of NO synthesis [6,7], although for both evidence is controversial [8,9]. Consequently, the mechanisms whereby the VEGF inhibitors exert their unfavourable vascular effects remains to be clarified.
In the present issue of the Journal, Cameron et al. used the venous occlusion plethysmographic technique to investigate the influence of bevacizumab on the forearm microcirculation, and assess its capacity to regulate the ET-1 and fibrinolytic activities in young healthy volunteers. The novel aspect of this elegant study is the demonstration that acute exposure to bevacizumab did not increase resting vascular tone and had no effect on agonist-induced endothelium-dependent relaxation. Moreover, bevacizumab did not influence endothelium-derived fibrinolytic factors or ET-1. The article by Cameron et al. is an interesting and well conducted report which, for the first time, explores the impact of an acute VEGF inhibitors infusion on the endothelial physiology and, more specifically, on the endothelium-derived products. Although reporting substantially negative data, which often are ignored or underestimated by the scientific community, the study provides intriguing information that encourages future research. Some aspects of this report deserve further considerations.
The first consideration concerns the rigorous methodology employed by the authors to explore the dynamic changes of endothelial-derived products induced by bevacizumab. By simultaneous arterial and venous blood sampling at the level of the forearm microcirculation (performed before and after a pharmacological stimulus), the technique employed by the authors allowed a simultaneous and accurate assessment of different aspects of the endothelial physiology, including the changes of endothelial function induced by the specific infusions, and the release of locally generated metabolites or uptake of externally infused substances. The combination of these two approaches, that is, the perfused-forearm model together with the assessment of net forearm uptake/release of fibrinolysis products is a gold-standard tool for the evaluation of different aspects of endothelial function and activity, as previously demonstrated .
A second consideration relates to a limitation of the study. In physiological conditions, vascular homeostasis is maintained by the opposite action of relaxing and contracting activities exerted by NO and ET-1, respectively. ET-1 acts by binding to two receptor subtypes, ETA and ETB, located on vascular smooth muscle cells and able to mediate the vast majority of ET-1 effects. ETB receptors are also found on endothelial cells, where their activation results in NO-mediated vasodilatation. In turn, NO counterbalances the contracting effect of ET-1. In their study, the authors utilized bradykinin, whose relaxant activity mainly derives from stimulation of the endothelium-derived hyperpolarizing factor (EDHF) , at variance from acetylcholine which induces vasodilatation predominantly via NO. The young healthy individuals recruited in the current study are characterized by a preserved NO availability, making the contribution of EDHF on endothelial physiology irrelevant. For these reasons, the utilization of acetylcholine together with a simultaneous NO synthase inhibitor (i.e. L-NMMA) could represent an appropriate pharmacological tool to gain further insights on the impact of VEGF inhibitor on NO/ET-1 axis. Acetylcholine infusion, with and without coadministration of the L-NAME, might have also provided a more precise assessment of the impact of bevacizumab on the NO/ET-1 balance, by documenting whether the pharmacological NO blockade might trigger a VEGF inhibitor-induced ET-1 release. By contrast, bradykinin is particularly useful to study endothelial function in different pathological conditions characterized by a reduced/absent NO availability, including essential hypertension, diabetes and hypercholesterolemia (and patients with active cancer as well), in whom EDHF is a relaxing compensatory mechanism to preserve endothelial function . On the contrary, these were not the patients investigated in the current study.
A third comment deals with one of the most important and frequent adverse/toxic effect of VEGF inhibitors, represented by BP increase . This effect may be due to pathophysiologic mechanisms and pathways not explored in the current study, such as microvascular rarefaction, increased intravascular oxidative stress and impaired natriuresis. Some of these phenomena are likely to result from longer exposure to VEGF inhibitors, thereby being outside the assessment provided by the acute setting of the current study. Others, however (changes in the local oxidative stress and vascular rarefaction), represent reversible alterations upon drug withdrawal , thus hypothetically representing functional phenomena that could be explored also in acute conditions.
In conclusions, the study of Cameron et al. is the first to explore, by a high-quality methodology, the relationship between bevacizumab, ET-1 and fibrinolysis in young healthy subjects. Along with this merit, the article encompasses a number of unresolved questions, including the exact role of NO and other endothelium-derived vasoactive pathways and oxidative stress on bevacizumab-mediated vascular damages, although with the limitations due to the acute experimental condition and the collection of data in a population which is not the target of this drug in real life. Future studies are warranted to better understand the mechanisms underlying the toxic effect of bevacizumab, achieve an earlier identification of patients at greater risk and develop drugs with an improved treatment tolerance.
Conflicts of interest
There are no conflicts of interest.
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