at all time-points) (Figure 14)

Figure 14: Mean arterial pressure (MAP) changes following intravitreal ranibizumab in 10 patients with neovascular age-related macular degeneration. There was no significant change in MAP during the period of follow-up (p> 0.05 at all time-points). Mean arterial pressure was 101 ± 9 mmHg at baseline, 100 ± 8 mm Hg at day 90 and 104 ± 10 mmHg at month 12.

Mean arterial pressure was 101 ± 9 mmHg at baseline, 100 ± 8 mm Hg at day 90 and 104 ± 10 mmHg at month 12. There was no significant change in IOP during the study period either (p> 0.05 at all time-points). Baseline mean IOP was 15.3 ± 2.8 mmHg at day 0, 15.8 ± 2.4 mmHg at day 90 and 16.8 ± 2.5 mmHg at month 12. No injection-related adverse events were observed throughout the follow-up period.

13. Discussion

A new era has emerged in the management of neovascular AMD with the advent of anti-VEGF agents. Both ranibizumab (Lucentis, Genentech/Roche) and bevacizumab (Avastin, Genentech/Roche), that are currently used in the treatment of ocular vascular diseases, are panisoform inhibitors of VEGF-A [256], therefore

impairing both pathological and physiological actions of VEGF-A. Some concerns may thus arise from blockage of all VEGF-A isoforms when multiple intravitreal injections of these agents are performed.

We have previously published our results on the short-term effect of IVT ranibizumab on the retinal arteriolar diameter in patients with neovascular AMD.[257] In the current study, we investigated the long-term effect of IVT ranibizumab on the retinal arteriolar diameter in patients with neovascular AMD. We found a significant decrease in retinal arteriolar diameter following each one of the 3 monthly IVT injections of ranibizumab with a mean decrease of 18.6 ± 7.2 % one month following the 3^rd injection when compared to baseline. This vasoconstrictory effect was maintained through month 12 with a 19.1 ± 8.3 % decrease of the retinal arteriolar diameter when compared to baseline following a median of 4 injections.

Moreover, this vasoconstrictory effect seems to be independent of the number of IVT injections of ranibizumab, as we found no correlation between number of injections and % diameter decrease at month 12, but this may be due to our small sample size.

There was no significant change in MAP during the period of follow-up, thus excluding an effect of systemic blood pressure fluctuations on the measured retinal vessel diameters.[258, 259] There were no significant changes in IOP either. Hence, neither systemic nor local factors other than IVT ranibizumab were likely to have influenced vessel diameter measurements.

In adults, VEGF acts at several levels in the vessel beds: it is a survival factor for endothelial cells, it increases microvascular permeability and induces vasodilation.[148] There is increasing evidence suggesting that the vascular effects of VEGF are mediated by NO production. Nitric oxide was originally discovered as an endothelium-derived relaxing factor and is known to be a potent vasodilator.[260] It has an important role in arteriolar tone maintenance [261] and retinal blood flow autoregulation by its vasodilatory effect.[262] An in vivo animal study, found that intravenous infusion of VEGF increased permeability in all ocular vasculatures, as well as ocular blood flow and mean arterial blood pressure, and that all permeability and hemodynamic changes induced by VEGF could be prevented by inhibition of NO synthase (Figure 15).[263] Several other reports also found that the VEGF-associated vascular permeability and hemodynamic changes are mediated by increased nitric oxide production.[264-268] Based on these observations, we can assume that the mechanism

by which ranibizumab was found to decrease retinal vessel diameter is inhibition of NO production.

Figure 15: VEGF interacts with nitric oxide to regulate vascular tone. The presence of VEGF is

associated with the production of nitric oxide. Nitric oxide is a messenger molecule (a molecule that carries signals between cells) that can regulate various physiologic functions, including vessel diameter thus retinal blood flow. Reducing nitric oxide production results in vasoconstriction; it has been hypothesized that this process could play a role in hypertension.[264, 269-273]

Apart from physiological actions, VEGF has other effects which, although triggered by pathological stimuli, are desirable. These include the capacity to promote the formation of collateral vessels, which is essential for recovery following ischemic events.[148, 274, 275]Impairment of collateral vessel development might be involved in cardiovascular ischemic events reported in trials using anti-VEGF treatment.[276]

Moreover, in central retinal vein occlusion (CRVO), restoration of venous flow is considered to occur either by recanalization of the central vein or the development of collateral vessels at the optic nerve head. In a study of 6 patients with CRVO of less than 3 month’s duration treated with repeated IVT injections of bevacizumab, collateral vessels did not develop in any patient after a mean follow-up of 12 months.[277]

Moreover, secretion of VEGF by RPE helps to maintain the choriocapillaris layer.[278-280] The choriocapillaris vasculature provides the outer retinal layers with oxygen and nutrients via intercellular junctions and endothelial cell fenestrations.

Atrophy of the choriocapillaris or loss of its fenestrations impairs nutritional support, which may lead to functional and morphologic damage of the RPE and photoreceptors

[279] with particular adverse consequences if the macular region is affected.

Intravitreal bevacizumab has been shown to significantly reduce choriocapillaris

endothelial cell fenestration in primate eyes.[281]

Vascular endothelial growth factor was so named because of initial observations demonstrating endothelial vascular cells as its target. However, VEGF may act on other cell types, based on the presence of either VEGFR-1 or VEGFR-2 on those cells. Increasing evidence points to a role for VEGF in neuronal cell growth and survival. In the eye, both neuronal and glial cells express VEGF receptors [282]

and VEGF and has a neuroprotective effect in the ischemic retina.[283] In this regard, VEGF-A is capable of protecting neurons from ischemia-reperfusion injury both by increasing blood flow to assist the damaged tissue and by directly promoting survival of neuronal cells.[283] Moreover, a dose-dependent decrease in ganglion cells was found following injection of an antibody that blocks all VEGF isoforms in rats.[283]

This may be particularly worrisome when considering to treat patients with diabetic retinopathy with anti-VEGF agents, as these patients demonstrate altered neuronal and glial functions early in the disease process.[284, 285]

These findings could have clinical implications, since the long-term neutralisation of retinal VEGF may have unintended consequences, including loss of neural retina cells and increased risk of circulation disturbances in the choriocapillaris.[286] Rouvas et al. reported a case of retinal angiomatous proliferation in a patient with AMD treated with a combination of photodynamic therapy (PDT) and an IVT injection of bevacizumab. They found, on indocyanine-green angiography, that the choroidal hypofluorescence related to the PDT spot extended beyond the area of the initial treatment spot size and was associated with deterioration in VA. They attributed this effect to enhancement of the photothrombotic effect of PDT on the normal choroidal vessels by blockage of VEGF related to administration of bevacizumab.[287]

Recently, the use of ranibizumab has been expanded to indications other than neovascular AMD. Pilot trials have indicated that intravitreal ranibizumab seems to be beneficial in patients with macular oedema secondary to diabetic retinopathy and retinal vein occlusions, at least in the short-term. [288-291] Similarly, bevacizumab is increasingly used in conditions other than AMD, including proliferative diabetic retinopathy, CNV complicating pathological myopia, neovascular glaucoma, and macular oedema due to diabetes, retinal vein occlusion, or uveitis. [292] With the

increasing use of ranibizumab and bevacizumab for VEGF-mediated ocular diseases, it is important to be aware of their effect on the normal retinal vasculature.

Soliman et al. studied 10 eyes of 10 patients with diffuse diabetic macular edema and found that there was a trend towards vasoconstriction following 3 IVT injections of bevacizumab.[293] The authors assessed the retinal vessel diameter changes at baseline and 1 month following the third IVT injection of bevacizumab using customized software on early phase FA. The lack of a significant effect of bevacizumab on retinal vessels’ diameter contrasts with that of another study using the same technique to measure retinal vessel diameter changes following IVT triamcinolone acetonide, which has also anti-VEGF properties; in 14 eyes with diabetic macular oedema, IVT injection of triamcinolone was followed 1 week later by a significant decrease of 10.8 % and 5.1% in the diameters of retinal veins and arteries, respectively.[294] A subsequent study also found a significant decrease in retinal arteriolar and venular diameter in eyes with diabetic macular oedema 3 months after a single IVT triamcinolone injection.[295]

Several investigators have recently reported the association of bevacizumab with ischemic retinal changes [296-299], suggesting a reduction of retinal blood flow following its intraocular administration. Kim et al reported a case of perfused CRVO conversion to ischemic CRVO 3 weeks following IVT bevacizumabKim K.S. et al and Yokomaya et al described a case of extensive occlusion of both retinal arteries and veins 4 weeks following intracameral bevacizumab in a patient with neovascular glaucoma and diabetic retinopathy.[299] Lee et al. reported a case of a patient with proliferative diabetic retinopathy who developed extensive and multiple retinal hemorrhages 1 week following IVT bevacizumab as an adjuctive treatment to pars plana vitrectomy.[298] The authors suggested that blockage of VEGF by bevacizumab induced further ischemic damage to the retina and development of hemorrhages. Chen et al described the case of a patient with non-proliferative diabetic retinopathy, who noted a significant decrease in visual acuity within 2 days of treatment with bevacizumab. Fluorescein angiography demonstrated an enlargement of the foveal avascular zone and persistent late leakage. The authors concluded that the use of IVT bevacizumab in chronic, refractory diabetic macular edema may cause acute visual acuity loss by disrupting an already fragile vascular perfusion status, leading to macular ischemia.[296] Similarly, Pieramici et al found increased macular ischemia in FA and increase in the extent of retinal hemorrhages in a patient with

perfused CRVO following repeated IVT injections of ranibizumab.[290] Contrary to these observations, other authors found no increasing ischemia following IVT bevacizumab for diabetic macular oedema.[300, 301] However, the presence of macular ischemia has a deleterious effect on the visual outcome after IVT bevacizumab in patients with macular oedema secondary to diabetes and branch vein occlusion despite improvement in central retinal thickness.[302, 303] Overall, these reports, suggest retinal circulatory disturbances as a consequence of the anti-VEGF treatment.

In summary, we showed that IVT ranibizumab decreases retinal arteriolar diameter in patients with neovascular AMD. A possible interpretation of our results is that the changes we measured in the retinal arteriolar diameter may have been influenced by the exudative macular degeneration itself and its associated manifestations, such as be macular edema, subretinal fluid and/or inflammation, with the reduction in retinal vessel diameter following IVT ranibizumab reflecting progressive return to the normal diameter due to reduction in macular edema, subretinal fluid and/or inflammation (which would be the result of VEGF being downregulated after anti-VEGF treatment) and subsequent recovery of the retinal function rather than a true vasoconstriction related to VEGF blockage itself.

Inversely, this hypothesis would also mean that macular edema, subretinal fluid and/or inflammation (resulting from upregulation of VEGF) would be responsible for a vasodilation at baseline before treatment in eyes with neovascular AMD. This hypothesis may be supported by the effect that we found a continuous reduction in mean retinal arteriolar diameter during the follow-up period which was independent of frequency of injections. Nevertheless, we found no association between retinal arteriolar diameter reduction and CNV activity, subretinal fluid or central retinal thickness. Moreover, the VEGF, though upregulated within the choroidal neovascular membranes and the RPE,[304, 305] is not increased in the vitreous cavity in patients with neovascular AMD compared to normal controls, so no effect on the retinal arteriolar diameter could have been present at baseline.[306, 307] In addition, there is no evidence either, in favor of an association between retinal vessel diameter and neovascular AMD. Population-based cohort studies have investigated the association of retinal vessel diameter with the incidence of early (indistinct soft or reticular drusen or combined distinct soft drusen and retinal pigmentary abnormalities) and late (geographic atrophy or neovascularization) AMD. The Beaver Dam Eye Study

showed that retinal arteriolar diameter was not related to the 10-year incident early or late AMD as defined above.[73] Similarly, the Rotterdam Study found no association between incident AMD and retinal vessel (artery or vein) diameter.[96] This latter finding is consistent with data from the Atherosclerosis Risk in Communities Study [97] and the Blue Mountains Eye Study.[98] More recently, the Singapore Malay Eye Study showed no significant association between retinal arteriolar caliber and neovascular AMD.[308]

In conclusion, we showed that intravitreal ranibizumab induces retinal arteriolar vasoconstriction in patients with neovascular AMD that persists up to one year after repeated injections. Considering their mechanism of action as mentioned above, we hypothesize that anti-VEGF agents lead to retinal arteriolar vasoconstriction directly, by blocking the production of NO which induces the vasoconstrictiry effect. According to the Pousseille law, significant changes of the arterial diameter may result to ischemic changes following the intraocular administration of anti-VEGF. This is the first report in the literature of the long-term effect of an anti-VEGF agent on the retinal arteriolar diameter. In order to do so, we used a RVA which is an accurate, fast and non-invasive method for evaluation of retinal vascular response and offers a unique opportunity for online measurements and estimation of diameter changes in retinal vessels.

In the current study, we did not calculate retinal blow flow changes but only retinal arteriolar diameter changes to intravitreal ranibizumab. However, retinal vessel diameter is a useful surrogate for retinal perfusion, with changes in the diameter of the retinal arterioles resulting changes in retinal capillary blood flow. Indeed, according to the Poiseuille’s law, flow is proportional to the fourth power of the tube diameter [309] and there are data indicating that, to a close approximation, the Poiseuille formula describes blood flow through the retinal arterioles.[310-313]

There are three primary factors that determine the resistance to blood flow within a single vessel: Vessel resistance (R) is directly proportional to the length (L) of the vessel and the viscosity (η) of the blood, and inversely proportional to the radius to the fourth power (r⁴). Of these three factors, the most important quantitatively and physiologically is vessel diameter. The reason for this is that vessel diameter changes because of contraction and relaxation of the vascular smooth muscle in the wall of the blood vessel. Furthermore, as described below, very small changes in vessel diameter lead to large changes in resistance. Vessel length does not change

significantly and blood viscosity normally stays within a small range (except when hematocrit changes). Because changes in diameter and radius are directly proportional to each other (D = 2r; therefore D∝r), diameter can be substituted for radius in the following expression.

Therefore, a vessel having twice the length of another vessel (and each having the same radius) will have twice the resistance to flow. Similarly, if the viscosity of the blood increases 2-fold, the resistance to flow will increase 2-fold. In contrast, an increase in radius will reduce resistance. Furthermore, the change in radius alters resistance to the fourth power of the change in radius. For example, a 2-fold increase in radius decreases resistance by 16-fold! Therefore, vessel resistance is exquisitely sensitive to changes in radius.

The relationship between flow and vessel radius to the fourth power (assuming constant ∆P, L, η and laminar flow conditions) is illustrated in the figure above. This figure shows how very small decreases in radius dramatically reduces flow.

This relationship (Poiseuille's equation) was first described by the 19th century French physician Poiseuille. It is a description of how flow is related to perfusion pressure, radius, length, and viscosity. The full equation contains a constant of integration and pi, which are not included in the above proportionality. In the body, however, flow does not conform exactly to this relationship because this relationship assumes long, straight tubes (blood vessels), a Newtonian fluid (e.g., water, not blood which is non-Newtonian), and steady, laminar flow conditions.

Nevertheless, the relationship clearly shows the dominant influence of vessel radius on resistance and flow and therefore serves as an important concept to understand how physiological (e.g., vascular tone) and pathological (e.g., vascular stenosis) changes in vessel radius affect pressure and flow.

Therefore, our findings suggest that IVT ranibizumab, by decreasing retinal arteriolar diameter, may reduce retinal capillary blood flow. Combination of continuous laser Doppler velocimetry[312] or blue field simulation technique [314]

with a RVA could provide real-time assessment of blood flow in retinal vessels.

Further studies with larger sample sizes are needed to confirm these results as well as potential adverse effects on the retinal circulation in patients with neovascular AMD and with retinal vascular diseases.

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