Influence of Ranibizumab on Vascular Endothelial Growth Factor Plasma Level and Endothelial Progenitor Cell Mobilization in Age-Related Macular Degeneration Patients: Safety of Intravitreal Treatment for Vascular Homeostasis

Abstract

Purpose:

Intravitreal ranibizumab, which neutralizes vascular endothelial growth factor (VEGF), nowadays constitutes the first-line treatment in neovascular age-related macular degeneration (AMD). However, its potential systemic effect on vascular homeostasis as the consequence of such therapy has not been extensively investigated.

Methods:

Peripheral blood (PB) samples from 12 patients with newly diagnosed neovascular AMD were analyzed before as well as 1 and 4 weeks after intravitreal treatment with ranibizumab. VEGF plasma levels, the number of circulating endothelial progenitor cells (EPCs), and the intracellular expression of hypoxia-inducible factor (HIF) in PB cells were determined by enzyme-linked immunosorbent assay, flow cytometry, and real-time quantitative reverse transcriptase–polymerase chain reaction assays, respectively.

Results:

No significant changes within the analyzed parameters were found in the first or fourth weeks after ranibizumab injection compared with the primary, basic values before treatment. Based on our findings, intravitreal ranibizumab does not induce significant systemic effects or vascular impairment.

Conclusions:

Evaluation of the VEGF plasma level, the PB EPC concentration, and intracellular HIF expression may be supportive indicators of drug safety for ranibizumab.

Introduction

Ranibizumab is a recombinant, monoclonal IgG1 antibody antigen-binding fragment (Fab) designed to bind and inhibit all active forms of human vascular endothelial growth factor (VEGF). Ranibizumab binds with high affinity to the VEGF-receptor binding site on multiple isoforms of VEGF such as VEGF₁₁₀, VEGF₁₂₁, and VEGF₁₆₅, thereby preventing the binding of VEGF to its receptors VEGFR-1 and VEGFR-2. All 3 isoforms were shown to stimulate human endothelial cell proliferation, whereas ranibizumab neutralized this proliferation in a dose-dependent manner.¹

Anti-VEGF antibodies, along with ranibizumab, nowadays constitute the first-line treatment in neovascular age-related macular degeneration (AMD). By intravitreal application of the drug, the dosage can be kept low while maximizing its effect on choroidal neovascularization and minimizing potential adverse systemic effects. Although the eye is relatively isolated from systemic circulation, the barrier is often disrupted in ophthalmic neovascular disorders. This means that despite being administered as an intraocular injection, the drug can be detected in circulating blood. The serum concentration after intravitreal administration of ranibizumab in cynomolgus monkeys was found to be more than 1,500-fold lower than the concentration in the vitreous body² and below the limit of quantitation in rabbits.³ In patients with neovascular AMD, after a monthly intravitreal administration, maximum ranibizumab serum concentrations were low (0.3 to 2.36 ng/mL). These levels were below the concentration of ranibizumab (11 to 27 ng/mL) thought to be necessary for inhibiting the biological activity of VEGF-A by 50%, as measured in an in vitro cellular proliferation assay.⁴

Nevertheless, systemic penetration suggests the availability of ranibizumab to inactivate VEGF in peripheral blood (PB), leading to the inhibition of vascular proliferation and the development of tissue ischemia. Therefore, monitoring plasma VEGF concentrations after intravitreal ranibizumab administration may provide an insight into its potential adverse systemic reactions. In parallel, measuring the level of circulating endothelial progenitor cells (EPCs) in blood now appears to be a viable strategy for assessing the reparative capacity of the vascular system. Accumulating evidence, mostly obtained during the past decade, suggests that EPCs possess the potential to proliferate and differentiate into mature endothelial cells. Upon stimulation they can be mobilized into peripheral circulation, home to ischemic sites, and promote vascular recovery and re-endothelialization of damaged areas.⁵ Thus, circulating EPCs play an important role in accelerating endothelialization in areas with vascular damage, and low circulating EPC levels have prognostic implications being associated with death from cardiovascular events.⁶ As EPCs express VEGF-R, it has been proven that their migration/homing undergoes regulation via the VEGF/VEGF-R axis.⁷ Hence, vascular homeostasis, which ultimately depends on VEGF expression and EPC availability in circulation, is maintained through a precise balance between these crucial parameters.

In this study, we sought to explore this issue by measuring VEGF plasma levels in patients with the neovascular form of AMD after single intravitreal injections of ranibizumab. Further, to determine its potential influence on EPC mobilization, we also quantified the number of circulating CD34⁺CD133⁺VEGR-2⁺, which were referred to as early endothelial progenitors in the abovementioned group of patients. Finally, to assess the influence of the employed therapy on tissue oxygen availability and indirectly on local metabolism, we analyzed intracellular expression of hypoxia-inducible factor (HIF) in PB cells.

Methods

Characteristics of the study groups

Twelve patients with newly diagnosed neovascular AMD who were treated at the Department of Ophthalmology of Pomeranian Medical University of Szczecin were enrolled in the study. All of the enrolled subjects underwent a complete ophthalmic examination, that is, visual acuity, intraocular pressure measurements, and dilated fundus examinations using slit-lamp biomicroscopy. In all cases, 30° color stereo fundus photographs of the macular region of both eyes were taken. The AMD phenotypes were characterized by fluorescein angiography as well as by optical coherence tomography imaging. All patients were scheduled to receive intravitreal injections of 0.5 mg/0.05 mL of ranibizumab. One eye was treated in each patient. All injections were performed under sterile conditions in an operation room following a standardized protocol. The PB was collected before the first, initial intravitreal injection, and subsequently 1 week and 1 month after the first ranibizumab administration. Medical history and smoking status data were collected from the patients, and special attention was paid to arterial hypertension and pre-existing cardiovascular and cerebrovascular conditions. The study adhered to the tenets of the Declaration of Helsinki and approval was obtained from the Local Research Ethics Committee. Moreover, each patient gave written informed consent for his or her involvement.

Blood sample collection

Venous blood was collected from all subjects in tubes containing EDTA as an anticoagulant. The plasma was separated by centrifugation (15 min, 2,000g, 20 °C) within 2 h after collection, divided into aliquots and frozen at −70 °C until the assays were performed. The red blood cells were lysed with the use of BD Pharm Lyse lysing buffer (BD Biosciencies) for 15 min at room temperature to isolate PB nuclear cells (PBNCs).

Laboratory assays

Enzyme-linked immunosorbent assays

Plasma concentrations of human VEGF were measured using commercially available enzyme-linked immunosorbent assays, Quantikine human immunoassays (R&D Systems) according to the manufacturer's protocol. Absorbance was read at 450 nm using an automated Microplate Reader EL_X 808 _IU (Bio-Tek Instruments Inc.). The results were analyzed using a log–log quadratic curve fit.

Flow cytometry

Two million (2×10⁶) PBNCs were stained with the monoclonal antibodies anti-CD34 conjugated with fluorescein isothiocyanate (FITC) (BD Biosciencies), anti-CD309 (VEGFR-2/KDR) conjugated with phycoerythrin (PE; BD Biosciencies), and anti-CD133 conjugated with allophycocyanin (APC; Miltenyi Biotec) for the analysis of CD34⁺CD133⁺VEGR-2⁺ EPCs. Samples stained with the appropriate isotype control, that is, IgG1-FITC (BD Biosciences, Pharmingen; clone MOPC-21) and IgG1-PE (BD Biosciences, Pharmingen; clone MOPC-21), and IgG1-APC (Miltenyi Biotec), were examined in parallel. The samples were incubated with antibodies for 30 min on ice, washed twice in phosphate-buffered saline, and subsequently fixed in 1% formaldehyde. Flow analysis was performed on an LSRII instrument (BD Biosciences). At least 2×10⁵ events were acquired and analyzed using BD FACSDiva software (BD Biosciences). The number of cells in each population was expressed as the percentage of the total events.

Real-time quantitative reverse transcriptase–polymerase chain reaction

Total mRNA was isolated from PBNCs (2×10⁶) with the RNeasy Mini Kit (Qiagen GmbH). The RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase and oligo dt primers (Fermentas International Inc.). Quantitative assessment of HIF mRNA levels was performed by real-time reverse transcriptase–polymerase chain reaction (PCR) on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad Inc.). A 25-μL reaction mixture contained 12.5 μL of iQ SYBER Green Supermix reagent, 10 ng of cDNA template, and 1 pair of the primers: 5′-ATCCATGTGACCATGAGGAA-3′ (forward) and 5′-CTCGGCTAGTTAGGGTACAC-3′ (reverse) primers for HIF or 5′-AATGCGGCA TCTTCAAACCT-3′ (forward) and 5′-TGACTTTGTCACAGCCCAAGATA-3′ (reverse) primers for beta-2 microglobulin (B2M), designed with Primer Express software (Applied Biosystems). The real-time cycling conditions were 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, 60°C for 1 min, and 72°C for 15 s. Relative quantification of endothelin-1 mRNA expression was performed with the comparative Ct method. The relative quantization value of the target, normalized to an endogenous control B2M (housekeeping) gene and relative to a calibrator, was expressed as 2^ΔCt, where ΔCt=[Ct of the endogenous control gene (B2M)] – [Ct of the target gene (HIF)].

Statistical analysis

The significance of changes in the parameters measured between the time points was determined using the Wilcoxon signed rank test. Associations between the level of PB EPCs, the plasma VEGF concentration, HIF expression, and the selected parameters (age, sex, smoking status, coexisting hypertension, and atherosclerosis) were evaluated using Spearman's rank correlation coefficient for age and the Mann–Whitney test for the other variables. A P value of P<0.05 was considered statistically significant.

Results

The characteristics of the patients are summarized in Table 1.

Table 1.

Clinical Characteristics of the Study Group

Number of subjects	12
Sex (male/female)	5/7
Patient's age (years, mean±standard deviation)	75.8±7.2
Smoking status
Current	20%
Past	50%
Hypertension	70%
History of ischemic heart disease or stroke	50%

We hypothesized that intravitreal ranibizumab might well reveal systemic effects in AMD patients via a direct influence on the VEGF plasma level and indirectly on the EPC concentration in PB, as well as having effects on local oxygen availability accessibility. However, in our study we did not find any significant differences in plasma VEGF levels in these patients before the intravitrael administration of ranibizumab (median: 49.34 pg/mL) or 1 week afterward (median: 52.57 pg/mL; P=0.31). Similarly, 4 weeks after ranibizumab delivery the VEGF levels were still stable without any significant changes compared with their basic, primary level (52.57 pg/mL; P=0.76; Fig. 1a).

FIG. 1.

(A) VEGF plasma concentrations before and after the administration of ranibizumab. (B) The percentages of EPCs circulating in the peripheral blood of AMD patients. (C) Expression of HIF mRNA in isolated PBNCs collected from patients enrolled in the study. The amount of the transcript is expressed in arbitrary units relative to the control gene B2M (2^ΔCt, where Ct represents the difference in threshold cycle between the control and the target gene). The results are presented as mean±SD. VEGF, vascular endothelial growth factor; EPCs, endothelial progenitor cells; AMD, age-related macular degeneration; HIF, hypoxia-inducible factor; PBNCs, peripheral blood nuclear cells; SD, standard deviation.

Next, we analyzed the concentration of circulating EPCs to explore another important parameter that is crucial for the control of vascular homeostasis. Figure 1b shows the concentration EPCs in PB of the study subjects. Accordingly, 1 week after ranibizumab administration the number of circulating EPCs was lower than before the injection (medians: 0.002% vs. 0.0013%, respectively); however, the difference was borderline significant (P=0.1). Moreover, 4 weeks after ranibizumab delivery the concentration of EPCs did not differ compared with the initial, basic level (median: 0.0013%; P=1.0).

Finally, we investigated the third key element, that is, the intracellular expression of HIF in PB cells, which represent a transcription factor that responds to changes in the availability of oxygen in the cellular environment, specifically to hypoxia. We found no significant differences in the HIF expression measured before the administration of ranibizumab (median: 0.04) compared with after intravitreal injection of the drug at either one (median: 0.05; P=0.86) or 4 weeks (0.04; P=0.37) afterward (Fig. 1c).

VEGF concentrations, intracellular HIF expression, and the number of circulating EPCs were unaffected by age, history of smoking, hypertension, or coinciding atherosclerosis.

Discussion

VEGF plays an important role in homeostasis of the cardiovascular system. It promotes vascular endothelial cell growth and angiogenesis in vitro and in vivo, as well as acting as a survival factor for endothelial cells, preventing their apoptosis and inducing re-endothelialization of damaged areas.⁸ Therefore, the use of VEGF inhibitors in AMD could theoretically be associated with an increased risk of cardiovascular events.

The safety profile of ranibizumab has been evaluated in several clinical trials.⁹ The main nonocular adverse systemic events during intravitreal anti-VEGF therapy of AMD patients are postulated to be thromboembolic events, although incidence rates in the published ranibizumab studies do not differ significantly between treated and untreated patients, nor are they significantly higher compared with the incidence rates in this age group.^10,11 However, data from the Safety Assessment of Intravitreal Lucentis for AMD (SAILOR) study indicates that there was a numerically higher incidence of stroke among the patients who received 0.5 mg (1.2%) ranibizumab compared with those who received 0.3 mg (0.7%) ranibizumab. The risk appeared to be higher in patients who had had previous stroke.¹²

The results of this retrospective analysis must be carefully evaluated and we cannot ignore the fact that the sample sizes of these studies were much smaller than are used in cardiovascular trials to confirm the benefits or adverse effects of new drugs.¹³ Thus, it is very difficult to draw valid conclusions about the cardiovascular safety of antiangiogenic therapy. There is some evidence to show that intravitreal anti-VEGF injections may result in systemic absorption, with the potential for injury to organs that are reliant on VEGF, such as the kidney. A nonimmune microangiopathic hemolytic anemia with schistocytes and thrombocytopenia has been reported in patients treated with ranibizumab after 4 intravitreal injections.¹⁴ Such a systemic cross-over would raise concerns about severe systemic complications after intravitreal treatment with ranibizumab. However, the few reports available in this field are substantially contradictory. While some case reports describe putative effects of intravitreally applied ranibizumab or bevacizumab in the untreated fellow eye, thereby implicating a significant cross-over of the drug into the systemic circulation,¹⁵ such a contralateral effect has not been observed by other authors.⁴

Hence, there is a growing need to elucidate the real impact of ranibizumab on vascular system homeostasis. To the best of our knowledge, published data are not available for VEGF concentrations in PB before and after ranibizumab in a human case series. Further, the data regarding the impact of locally administrated ranibizumab on the systemic mobilization of EPCs and HIF expression are lacking. Therefore, we undertook a precise evaluation of this essential issue to provide a novel insight into the mechanism of the potential systemic action of the drug. In this study, we found no significant change in VEGF plasma levels and no disturbances in EPC mobilization or HIF expression in PB cells after a single dose of ranibizumab at the 1 month follow-up. These results indicate that intravitreally injected ranibizumad might be safe since it did not induce detectable systemic changes in VEGF plasma levels, circulating EPCs, or HIF expression.

Interestingly, Matsuyama et al. reported that intravitreally injected bevacizumab in patients with type 2 diabetes resulted in a significant decrease in the levels of systemic VEGF.¹⁶ This indicates that intravitreally administrated bevacizumab does not only penetrate the retina, choroid, and intraocular blood vessels, but it also enters the general blood circulation. It is reasonable to assume that ranibizumab could be removed from the systemic circulation faster than bevacizumab due its smaller molecules (48 kd compared with 149 kd for bevacizumab); thus, it would not significantly influence VEGF plasma levels and subsequently has less pronounced systemic effects.¹⁷ Moreover, the possibility that the blood–retinal barrier might be more severely damaged in the course of diabetic retinopathy cannot be excluded; this would facilitate the effective penetration of the drug into the systemic circulation, and its substantially greater interaction with VEGF in PB.

Further, we cannot exclude the possibility that responsiveness to ranibizumab could differ between individuals. Patients who have lower systemic VEGF or EPC levels might well have lower thresholds of tolerance to anti-VEGF agents and would be at a higher risk of systemic atherosclerotic complications that could result from VEGF inhibition.¹⁸ Other, not yet precisely defined, pharmacokinetic or genetic factors should also be taken into consideration to explain the entire pathophysiological background.

In conclusion, based on our findings, intravitreally injected ranibizumab does not induce significant systemic effects or vascular impairment. Since placebo-controlled trials would be unethical because of the positive effect on vision loss demonstrated by ranibizumab, the evaluation of VEGF plasma levels and EPC concentrations in PB may be supportive indicators of the drug safety. As patients with AMD constitute a high-risk population for cardiovascular events, the safety of anti-VEGF therapies must be precisely and thoroughly assessed.

Footnotes

Acknowledgment

This work was supported by the Polish Ministry of Science and Higher Education (grant number: N N402 172137 to AM).

Author Disclosure Statement

None of the authors has a proprietary interest in the published data.

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