This study provides detailed information regarding VEGF induced direct vasoactive changes in both normal tone and pre-contracted retinal arterioles and demonstrates that VEGF exhibits vasoactive effects on porcine retinal arterioles. It has been strongly evidenced that the vasoactive properties induced by VEGF in retinal arterioles are clearly depend on the concentration and vessel conditions before the application of VEGF. The results also show that bevacizumab can inhibit the VEGF induced vasodilatations in pre contracted retinal arterioles. These results may be important not only to illuminate and interpret some consequences from patients treated by anti VEGF agents, but also to provide some possible clues as to how to avoid some side effects.
Anti-VEGF agents have been extensively used in ocular diseases in order to inhibit retinal neovascularisation [13
In considering therapeutic benefits and side effects of VEGF, it is important to bear in mind that VEGF has multiple physiological and pathological roles, and multiple factors could be influenced after modulation of VEGF activity at the systemic or individual organ level. In ocular neovascularisation and ischemic diseases, we need to consider changes in angiogenesis and permeability, and also blood flow. It has been reported that intravitreal ranibizumab could induce retinal circulation disturbances in patients with neovascular age-related macular degeneration [27
], neovascular glaucoma [29
], retinal vein occlusion [30
], and diabetic retinopathy [31
]. It is important to determine VEGF induced vasoactivity on retinal arterioles before addressing the possible effects of vitreal bevacizumab injection on the retinal circulation. Our results obtained from retinal arteriole are remarkably different than that from previous reports in other organs. Our results show that the vasoactive properties induced by VEGF in vessels without pre-contraction appear to be mildly contractile at low concentrations (~5% reduction in the vessel diameter) and less contractile at higher concentrations of VEGF (almost reaching the original diameter). However, the results obtained from other organs show dilatation responses of arteries, for example coronary arteries [3
], placental lobule [32
], uterine arteries [20
], and pulmonary arteries [21
]. One possible explanation is a difference in vasoactive properties between the retinal arteriole and peripheral vessels that were used in most of the previous studies. Retinal blood vessels are not innervated [33
]. Retinal arteriole tone is mainly regulated by local factors. The mediators derived from the endothelial cells and retinal tissue can be considered as local regulators in addition to physical and metabolic influences [38
]. However, the effective concentration levels in most previous studies are similar to that in our study indicating that vasoactive effect starts at concentrations as low as ~10-12
To our knowledge, the effect of VEGF in pre-contracted vessels has not previously been studied. Our results demonstrate that in ET-1 pre-contracted vessels, VEGF induced a potent concentration dependent vasodilatation response which was inhibited by bevacizumab at a similar concentration to that used clinically. The rationale of studying the effect of VEGF on pre-contracted vessels is that the majority of patients treated by bevacizumab are aged, and may have cardiovascular disease and increased vessel tone. Furthermore, VEGF levels in most ocular neovascular diseases are increased. Bevacizumab is a full-length, humanized monoclonal antibody directed against all the biologically active isoforms of vascular endothelial growth factor (VEGF-A). It could be expected that bevacizumab may modulate the retinal vessel tone. Our results demonstrated that bevacizumab can inhibit VEGF induced vasodilatation if the vessel is pre-contracted. Retinal ischemia in some patients may therefore be exacerbated by VEGF inhibition.
Since 2005 the experience with bevacizumab and other anti VEGF agents in ophthalmology has accumulated rapidly. Therapeutic benefits in neovascular ocular diseases have been demonstrated. However, despite the excitement, a number of critical issues have been raised. Two major issues need to be urgently addressed. Firstly, regrowth of new vessels is often found and permanent regression of neovascularization is rare, requiring multiple injections of anti VEGF agent. Secondly, associated ischemia after application of anti VEGF agents has been found in various neovascular ocular diseases. These issues may be interlinked. Neovascularisation in the eye and other organs occurs by both vasculogenesis and angiogenesis. It is now known that vasculogenesis occurs in the adult as endothelial precursor cells derived from the bone marrow enter the circulation in response to ischemic injury. In addition, hypoxia regulated factors are the key mediators of endothelial precursor cells and resident endothelial cells [11
]. Ideal treatments of neovascular ocular diseases should not only target reduced neovascularisation and vascular permeability, but also relieve the retinal ischemia/hypoxia if possible. The retina is a functionally active tissue, with arguably the highest oxygen demand tissue per weight, yet the retina has a very limited blood supply from the retinal circulation [40
]. Recently, reduced retinal perfusion has been demonstrated following anti VEGF treatment in eyes with branch retinal vein occlusion [45
]. Anti VEGF treatment further reduces retinal perfusion causing more severe ischemia/hypoxia which could be the cause of the regrowth of new vessels. The potential long-term effects of anti VEGF induced vasoconstriction may need to be considered.
An important question needing to be addressed is how to avoid the associated ischemic retinal and choriocapillaris changes after intraocular administration of anti VEGF agents [27
]. Our results show that bevacizumab is able to inhibit VEGF induced potent vasodilatation. It is possible that the anti-VEGF agent, bevacizumab may inhibit the vasodilatation by VEGF after vitreous injection. As a consequence, retinal blood flow could be reduced. Our results also show that VEGF induced vasodilatation only occurs in ET-1 pre contracted vessels. It could be assumed that in the cases in these clinical reports, VEGF may act as a vasodilator that counterbalances contractile vessel effects caused by the original disease before anti-VEGF injection. However, anti-VEGF agent could inhibit VEGF induced vasodilatation, and as consequence, vasoconstriction could be present. It is well known that VEGF has several family groups but it is the VEGF-A family that is principally involved in the regulation of vasoactive tone. VEGF-A has at least seven splice variants [46
]. It could be favourable if an anti-VEGF agent could be chosen that is not involved in the regulation of vasoactive tone, or better still, one that can potentially improve retinal blood flow. The design of a new anti VEGF agent should also consider the differences of vasoactive properties between the retinal vasculature and that in other organs, as well as between normal tone and pre-contracted vessels. In addition, the concentration of anti-VEGF agent also needs to be considered, and case selection may be important. Some cases in which ischemia is predicted, such as ischemic central retinal vein occlusion, added caution in the use of anti-VEGF treatment may be warranted in order to avoid exacerbating retinal ischemia.
There are undoubtedly complex mechanisms regulating ocular and serum levels of VEGF in health and disease. The increasing use of anti-VEGF drugs to treat retinal and choroidal neovascularisation, should be accompanied by further studies of VEGF induced changes in vascular tone in order maximise the therapeutic benefits and to minimise unwanted side effects.