Circulating endothelial and progenitor cells in age-related macular degeneration

Abstract

Purpose:

To evaluate circulating endothelial and circulating progenitor cells as biomarkers in age-related macular degeneration patients (both exudative and atrophic forms) in order to establish the possible clinical implication of their assessment.

Methods:

We have enrolled 44 age-related macular degeneration patients: 22 patients with a recently diagnosed exudative (neovascular) form (Group A) and 22 patients with an atrophic (dry) form (Group B). The control group consisted of 22 age and sex-matched healthy subjects (Group C). The number of circulating endothelial progenitor cells (CD34+/KDR+, CD133+/KDR+, and CD34+/KDR+/CD133+), circulating progenitor cells (CD34+, CD133+, and CD34+/CD133+), and circulating endothelial cells were determined in the peripheral venous blood samples by flow cytometry. Neovascular age-related macular degeneration patients were evaluated at baseline and 4 weeks after a loading phase of three consequent intravitreal injections of ranibizumab.

Results:

Comparing age-related macular degeneration patients with the control group, endothelial progenitor cell and circulating progenitor cell levels were not significantly different, while age-related macular degeneration patients showed significantly higher levels of circulating endothelial cells (p = 0.001). Anti–vascular endothelial growth factor treatment with intravitreal ranibizumab was associated with a significant reduction of endothelial progenitor cell levels, with no significant influence on circulating progenitor cells and circulating endothelial cells.

Conclusion:

We reported higher levels of circulating endothelial cells in age-related macular degeneration patients in comparison with the control group, thereby supporting the hypothesis of an involvement of endothelial dysregulation in the age-related macular degeneration and a reduction of the endothelial progenitor cell level in neovascular age-related macular degeneration patients after three intravitreal injections of ranibizumab.

Keywords

Introduction

Age-related macular degeneration (AMD) is the leading cause of vision loss in people over 50 years in developed countries. Its incidence and prevalence are increasing due to the progressive aging of the population.^1,2 There are two forms of the disease: an atrophic form—dry AMD (drAMD)—and a wet form—neovascular or exudative AMD (nvAMD). The latter occurs in approximately 10% of AMD patients and is characterized by pathological choroidal neovascularization (CNV), which develops from the choriocapillaris due to an imbalance between pro-angiogenic and anti-angiogenic factors.³ A process noted as vasculogenesis, not only restricted to embryogenesis, contributes in part to the formation of new blood vessels in an adult.⁴ The progenitor cells (circulating progenitor cells (CPCs) and endothelial progenitor cells (EPCs)) from bone marrow, in response to different types of stimuli (one of the most important is vascular endothelial growth factor (VEGF)), are directed toward the peripheral sites of neovascularization throughout the bloodstream and may develop into mature endothelial cells (ECs) in situ.^5–8 These cellular types not only contribute to the neo-angiogenesis in vivo and to the maintenance of the homeostasis of vascular endothelium but also participate in pathological processes.^9,10 Following the first studies conducted on experimental animal models, different authors have reported that bone marrow–derived progenitor cells participate as a source of endothelial, macrophage, and smooth muscle-like cells in CNV and the VEGF determines the recruitment of EPCs at a high rate into active choroidal and retinal neovascularization.^11–15 The number of progenitor cells in the peripheral blood cells, recruited in relation to CNV activity, and their possible role have been evaluated in a small number of clinical studies, which enrolled a small number of AMD patients using different methodologies for the identification of EPC. Furthermore, the association between circulating endothelial cells (CECs) as marker of endothelial damage in the AMD has been poorly investigated.^16–18 The main therapeutic approach for CNV is based on anti-angiogenic drugs that are injected into the vitreous. This treatment is able to block the progression of the disease, but does not restore visual function.^19,20 The response to the anti-angiogenic drugs in nvAMD patients may be highly variable. Some patients do not respond to treatment and others require further injections due to a partial response or a relapse.²¹ Furthermore, intravitreal injections can cause side effects and are costly.^22,23 The identification of biomarkers that allow early detection of ocular angiogenesis and treatment monitoring may be important as a clinical tool for the clinical management of patients with exudative AMD. In this study, we have evaluated certain cell populations in peripheral blood (EPCs, CPCs, and CECs) as potential biomarkers of AMD.

Methods

The study followed the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of the local institution, and all participants gave informed consent. In our study, we included AMD patients divided into two groups. Group A included naïve nvAMD patients and Group B included drAMD patients. A control group, labeled Group C, included age-matched control subjects. All patients were recruited from the Eye Clinic of Careggi Teaching Hospital, University of Florence. The inclusion criteria were an age of 50 years and diagnosis of AMD in the study eye. Regarding nvAMD, the first time-occurrence of sub-foveal CNV was taken into consideration. The exclusion criteria were presence or history of retinal diseases other than AMD in the study eye and previous treatments. Patients with refractive error higher than 3 sph or 1.5 cyl dpt were not included. None of the patients were receiving treatment for glaucoma or other significant eye diseases. Some of the ocular patients had only a slight clouding of the lens, stable in the 3-month follow-up.

All patients underwent a comprehensive ophthalmological examination including measurement of Snellen best-corrected visual acuity (BCVA), biomicroscopy of the anterior segment, Goldmann tonometry, and fundoscopy after dilatation with tropicamide 1% eye drops. The retinal posterior pole was studied by spectral-domain optical coherence tomography (SD-OCT; Topcon OCT MARK II) and using fluorangiographic technique (FA; Zeiss FF 450Plus retinal IR), with frames taken at 10 min after injection of the tracer. The diagnosis of AMD was made on symptoms reported by the patient, the retinal changes detectable on physical and instrumental examination of the fundus.

For all patients, we collected a detailed history of physiological and pathological conditions with particular attention to cardiovascular risk and medications. In particular, the pharmacological classes considered were angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, diuretics, beta-blockers, calcium channel blockers, statins, fibrates, and aspirin. The subjects were classified as having hypertension and diabetes according to the guidelines of the European Society of Hypertension/European Society of Cardiology and to those of the American Diabetes Association, respectively.^24,25 Dyslipidemia was defined according to the criteria of the ATP III Expert Panel of the US National Cholesterol Education Program.²⁶

Investigation protocol

We performed an observational, cross-sectional, prospective study. At baseline (t0), we collected a blood venous sample from Groups A, B, and C in order to determine the number of cells considered in the study (CPC, EPC, and CEC). Group A patients after the active CNV diagnosis underwent three intravitreal injections of ranibizumab carried out approximately 1 month after each other.

Patients from this group (Group A) underwent two evaluations, the first at baseline (t0) and the second at approximately 1 month after the third ranibizumab injection. Standard eye examination, OCT examination, FA and a venous blood sampling for CPC, EPC, and CEC quantification, respectively, were carried out at the two time points (t0 and t1). Two patients did not agree to the second blood collection.

Blood collection

Blood samples were obtained in the morning after an overnight fasting for each patient. They were collected from the antecubital vein into evacuated plastic tubes (Vacutainer) containing 0.17 mol/L ethylenediaminotetracetate (EDTA) for EPC, CPC, and CEC evaluation. No anticoagulant was used for obtaining sera samples. The samples were centrifuged at 2000g for 10 min at 4°C and then stored in aliquots at −80°C until analysis.

Flow cytometric analysis

EPC and CPC evaluation

CPCs and EPCs were assessed contemporarily using flow cytometry as previously described.²⁷ EPCs were identified through their expression of CD34, KDR, and CD133. CPCs were defined as cells forming a cluster with low side scatter, low-to-intermediate CD45 staining, and positive for CD34, CD133, and CD34⁄CD133²⁷ (Figures 1 and 2).

Evaluation of CECs

For each patient, 200 μL of whole blood anti-coagulated with EDTA was incubated with an aliquot of 10 μL of (1) anti-CD61—monoclonal antibody conjugated with allophycocyanin (PCA), (2) anti-CD31—monoclonal antibody conjugated with fluorescein isothiocyanate (FITC), (3) anti-CD45—monoclonal antibody conjugated with allophycocyanin and cyanine 7 (APC-Cy7), and (4) anti-CD146—monoclonal antibody conjugated with phycoerythrin (PE). Samples were incubated in the dark at room temperature for 15 min, and at the end of the incubation, they were lysed with the addition of 2 mL of NH₄Cl. In the gate of white blood cells, we acquired 500,000 events. CECs were defined as cells CD61 and CD45−, with morphological characteristics of CECs and with a double positivity for CD146 and CD31 (Figure 3).

Statistical analysis

Statistical analysis was performed using SPSS version 20.0 (Statistical Package for Social Sciences, Chicago, IL, USA) software for Mac. The results are expressed as mean ± standard deviation for parametric data, and as median and range for non-parametric data. The Mann–Whitney test for unpaired data was used for comparison between groups. The Wilcoxon test for non-parametric paired data was used to evaluate the differences in EPC, CPC, and CEC between time t0 and time t1. The correlation between age, body mass index (BMI), and number of different cell subtypes (EPC, CPC, and CEC) was assessed using the Spearman test for non-parametric data. Categorical variables were compared between groups using the Fisher exact test. A p-value < 0.05 was chosen as the cut-off for statistical significance.

Results

We have studied 44 AMD patients (15 males and 29 females, with an average age of 80 years) and 22 control subjects.

In Group A (22 patients: 7 males and 15 females with an average age of 83 years (interquartile range: 80–85 years)), we studied patients with a first diagnosis of active sub-foveal CNV due to AMD; of these, 20 patients had classic CNV and two patients occult CNV. The SD-OCT examination showed the presence of a localized iper-reflected thickening in correspondence to the complex EPR-Bruch’s membrane-choriocapillaris, attributable to the presence of CNV, and with intra-retinal fluid and an increase in the foveal thickness. At the first control (t0), BCVA average in the affected eye was between 2/10 and 8/10 with an average value of 3.7/10 (0.43 LogMAR) ± 2.10. After the treatment with ranibizumab in the affected eye, BCVA average was 3.9/10 (0.41 LogMAR) ± 1.94. Group B (22 patients, 8 males and 14 females, average age of 79 years (IQR: 7–84 years)) included patients presenting an atrophic maculopathy. The average BCVA at the time of the initial examination was between 1/30 and 8/10 in OO with an average value of 4/10 (0.40 LogMAR) ± 3.61. The OCT examination showed thinning of the neuroepithelium due to the absence of the outer retinal layer, and the loss of the retinal pigment epithelium (RPE), which leads to a reverse shadowing effect. Group C (control group: 22 patients, average age of 80 years (IQR: 78–84 years)) included patients that did not suffer from ocular or systemic diseases.

The prevalence of cardiovascular risk factors and of drugs known to influence the number of EPCs, CPCs, and CECs in nvAMD, drAMD, and control group are shown in Table 1. No significant differences in terms of clinical and demographical characteristics are found among the three groups (Table 1).

Table 1.

Clinical and demographic characteristics of AMD patients and control group.

	Control groupGroup C (n = 22)	nvAMDGroup A (n = 22)	drAMDGroup B (n = 22)	p for trend
Age (years), median and interquartile range	79 (73–84)	83 (80–85)	79 (73–84)	0.052
Males, n (%)	8 (36.4)	7 (31.8)	8 (36.4)	0.935
Hypertension, n (%)	10 (45.5)	12 (54.5)	15 (68.2)	0.311
Smoking habit, n (%)	2 (9.1)	5 (22.7)	5 (22.7)	0.352
Diabetes, n (%)	2 (9.1)	5 (22.7)	4 (18.2)	0.466
Dyslipidemia, n (%)	4 (18.2)	6 (27.3)	8 (36.4)	0.400
Family history of CAD, n (%)	2 (9.1)	4 (18.2)	4 (18.2)	0.005
Current therapy
ACE inhibitors	5 (22.7)	8 (36.4)	5 (22.7)	0.503
Anti-angiotensin II	0 (0)	7 (31.8)	5 (22.7)	0.068
Diuretics	1 (4.5)	4 (18.2)	1 (4.5)	0.192
beta-Blockers	1 (4.5)	4 (18.2)	1 (4.5)	0.597
Calcium channel blockers	1 (4.5)	0 (0)	3 (13.6)	0.155
Statins	4 (18.2)	4 (18.2)	6 (27.3)	0.696
Fibrates	0 (0)	1 (4.5)	0 (0)	0.362
Aspirin	0 (0)	5 (22.7)	4 (18.2)	0.067
Oral anticoagulants	0 (0)	1 (4.5)	2 (9.1)	0.351

AMD: age-related macular degeneration.

Evaluation of EPC, CPC, and CEC in AMD patients and in control subjects

No significant differences of EPC and CPC were observed between healthy subjects (n = 22) and AMD patients (n = 44). Post hoc analyses showed that, in healthy subjects, EPC and CPC were not significantly different from those found both in nvAMD and drAMD patients. Similarly, no significant difference in EPC and CPC between nvAMD and drAMD was found (Table 2). Regarding CEC, we found higher levels of CEC in both AMD groups (n = 44) than in healthy subjects (p = 0.001). Post hoc analyses demonstrated that both nvAMD and drAMD had a significantly higher number of CECs than controls, whereas no significant difference was found between nvAMD and drAMD (Table 2).

Table 2.

EPC, CPC, and CEC count in AMD patients and in control group.

Patient group	CEC	CPC			EPC
		CD34+	CD133+	CD34+/CD133+	CD34+/KDR+	CD133+/KDR+	CD34+/CD133+/KDR+
Control–group (Group C)	0 (0–1)	450 (282.5–565)	445 (280–564.5)	421.5 (270.75–507)	1 (0–2.25)	1 (0–2.25)	1 (0–2.25)
nvAMD patients (Group A)	1 (0–2)*	416 (319.75–569.5)	396.5 (317.5–539.25)	326.5 (238–518.25)	1.5 (0–3.25)	1.5 (0–3.25)	1 (0–2.5)
drAMD patients (Group B)	2 (0.75–2.25)**	402 (264–580.75)	396 (216–496.25)	306 (154.5–406.75)	1 (0–2.25)	0.5 (0–2)	0 (0–2)

EPC: endothelial progenitor cell; CPC: circulating progenitor cell; CEC: circulating endothelial cell; AMD: age-related macular degeneration.

p = 0.02, nvAMD patients versus control subjects.

p = 0.001, drAMD patients versus control subjects.

EPC, CPC, and CEC according to the presence of cardiovascular risk factors

We evaluated the number of EPCs, CPCs, and CECs according to the presence of traditional cardiovascular risk factors in AMD patients (44 patients) and control subjects (22 patients).

In the control subject group (Group C), dyslipidemic patients had a significantly lower number of EPCs (CD34+/KDR+, CD133+/KDR+, and CD34+/CD133+/KDR+) than non-dyslipidemic subjects (0 (0–0.5)/10⁶ cells vs 1 (0–3)/10⁶ cells, p = 0.026). This association was not found either in Group A or in Group B.

No significant correlation between CPC, EPC, CEC, and age was observed either in controls or in AMD patients (p > 0.1, the Spearman coefficient value has been reported in Supplementary Material Table 4). There were no significant differences in the number of EPCs, CPCs, and CECs according to gender, hypertension, diabetes, smoking habit, and family history of coronary artery disease (p > 0.1 for all comparisons using the Mann–Whitney test for unpaired data).

EPC, CPC, and CEC according to current treatment

In AMD patients, we studied the effect of different therapies on the number of EPCs, CPCs, and CECs.

AMD patients (44) treated with ACE inhibitors (13 patients) had a significantly higher number of CPCs than patients who were not on ACE-inhibitor treatment (CD34+: 583 (460–806)/10⁶ cells vs 384 (282–510)/10⁶ cells, p < 0.05; CD133+: 500 (450–775)/10⁶ cells vs 360 (278–452)/10⁶ cells; and CD34+/CD133+: 495 (322.5–649)/10⁶ cells vs 304 (182–398)/10⁶ cells, p < 0.05). On the contrary, treatment with angiotensin II receptor antagonists is significantly associated with a lower number of CPCs (CD34+: 355 (226.3–410.8)/10⁶ cells vs 478.5 (326–597.3)/10⁶ cells, p < 0.05; CD133+: 320 (218–398.5)/10⁶ cells vs 451 (322.5–526.3)/10⁶ cells; and CD34+/CD133+: 276 (153.5–380)/10⁶ cells vs 357.5 (247.5–495)/10⁶ cells, p < 0.05). Treatment with diuretics, beta-blockers, calcium channel blockers, statins, fibrates, and aspirin did not influence the number of cells (CPC, EPC, and CEC) (p > 0.1 for all comparisons using the Mann–Whitney test for unpaired data).

Evaluation of the CPC, EPC, and CEC in relation to anti-VEGF treatment in the exudative AMD patient

EPC, CPC, and CEC levels in different groups are shown in Table 3. Two nvAMD patients were not available for the second blood test after anti-VEGF injections. In nvAMD patients, a loading phase of three consecutive intravitreal injections of ranibizumab was associated with a significant reduction of the EPC number, whereas no significant influence on CPCs and CECs was observed. At t1, nvAMD patients had higher, but not statistically significant, levels of EPCs and CECs in comparison with healthy controls, whereas CPCs did not significantly differ between patients and controls (Table 3).

Table 3.

Effect of anti-VEGF treatment in the exudative AMD patients.

	Pre-treatment (n = 20)	Post-treatment (n = 20)	p
CEC	1 (0–2)	1 (0–1.8)	0.718
CD34+	416 (319.8–569.5)	430 (264–532)	0.313
CD133+	396.5 (312.5–557.8)	438 (237.3–498.5)	0.370
CD34+/CD133+	313.5 (218–524.8)	326.5 (238–518.3)	0.322
CD34+/KDR+	1.5 (0.3–2.8)	0 (0–1)	0.020
CD133+/KDR+	1.5 (0–3.0)	0 (0–1)	0.011
CD34+/CD133+/KDR+	1.0 (0–2.0)	0 (0–1)	0.038

VEGF: vascular endothelial growth factor; AMD: age-related macular degeneration; CEC: circulating endothelial cell.

Discussion

In a group of AMD patients, we investigated the role of CPCs and CECs as possible biomarkers for the management of patients affected by AMD. We found the following:

AMD patients, both the exudative and atrophic forms, had significantly higher levels of CECs than healthy subjects, whereas no significant differences in the CPC and EPC number were observed among the three groups.

Treatment with anti-VEGF (ranibizumab) of nvAMD is associated with a significant reduction of the EPC number, but not with the CPC and CEC number.

The endothelium is a major regulator of local vascular homeostasis. In fact, ECs synthesize several factors in response to various cytokines as well as physical and chemical stimuli.²⁸ A certain role of the endothelial dysfunction was demonstrated in AMD.²⁹ Our results demonstrated that the number of the CEC was significantly higher in AMD patients than in control subjects, independent of the type of AMD. This is an interesting result, which confirms and extends to drAMD the observation derived from Machalinska et al.’s study,¹⁷ showing that nvAMD is characterized by a higher number of CEC than age-matched controls.

An increased CEC count represents a novel marker of endothelial damage,³⁰ as CEC are mature cells that have detached from the vessel wall resulting from apoptosis in response to inflammatory and oxidative stress, alteration of endothelial and sub-endothelial cellular adhesion molecules, sub-endothelial matrix proteolysis, and mechanical as well as drug-induced endothelial damage.³⁰ In our study, we failed to detect in AMD patients a significant association between EPC, CPC, cardiovascular risk factors, and drug use. These observations may be ascribed to the advanced age of our patients, as it is well-known that in advanced age, the number of progenitor cells is drastically reduced, therefore the contemporary presence of other cardiovascular risk factors cannot further reduce the EPC and CPC number. Furthermore, the mechanisms underlying the balance between the speed/frequency of progenitor cell mobilization from bone marrow, their increased engraftment, and their turnover at the sites of vascular injury are still unknown.

Our results do not find any significant difference in the EPC and CPC numbers in AMD patients, both exudative and atrophic. These data are consistent with those obtained by Say et al.’s study³¹ which failed to demonstrate a significant role for EPC in nvAMD when EPC were assessed by fluorescence-activated cell sorting (FACS), whereas the authors found a higher number of EPCs in nvAMD than in non-nvAMD patients when these cells were assessed by another methodology (the automated rare cell analysis (ARCA)).³¹ These results showed the importance of methodologies used to identified and evaluate EPC.

In contrast with our study, two studies^17,32 using cytofluorimetric analysis, found a significant difference in the EPC number between nvAMD patients and healthy subjects: the first study detected a higher number of EPCs in AMD patients than in controls, whereas the second study showed that the EPC number was significantly lower in active AMD than in healthy controls. These discrepancies may be ascribed to the different definition of EPC, because in the first study EPC were defined as CD34+/CD133+/CD144+ cells and in the second study as CD34+/CD133+/VEGFR2 (KDR)-positive cells. Circulating EPC express, with different intensity, a variety of markers that are typical for the endothelial lineage, including CD144 which binds to the vascular endothelial (VE)-cadherin receptors. However, it has been shown that VE-cadherin may identify cells in a more advanced stage of maturation along the endothelial differentiation process.³³ The evaluation of the activities of EPC, that is, the capability of colony-forming units, provided contrasting results,^18,32,34 due to the different methodologies used. These conflicting results highlight the need to clearly define the methodology for evaluating EPC, in order to determine the timing of EPC assessment in relation to the phases of disease activity, and to extend the study to a higher number of AMD well-characterized patients since the majority of the studies had investigated few AMD patients.

Concerning the role of CPC in AMD, in our study, we did not detect any significant difference among nvAMD, drAMD, and controls. There is only one clinical study in the literature showing a CPC (CD34+ cells) increase in patients with active CNV compared with those with stable CNV and control subjects. Furthermore, the functions of circulating EPC (CFU-EC and migration) have been shown to decrease in patients with larger or bilateral CNV involvement,¹⁶ suggesting that an active CNV lesion may signal mobilization of CD34+ cells from the bone marrow into the peripheral circulation and that CD34+ cells might protect rather than promote CNV formation, as previously suggested.³⁵

Our protocol study included treatment of exudative AMD patients with anti-VEGF drugs. We found that this treatment is associated with a significant reduction of EPC, but not of CPC and CEC. Previous experimental models of CNV have demonstrated that bone marrow–derived EPC represent about 50% of the cells incorporated into the new vessels and that the prevention of EPC incorporation reduced the size of the CNV lesion.^11,36 The intravitreal injections of anti-VEGF in nvAMD are associated with a beneficial effect in terms of the reduction of angiogenesis, thus supporting the role of EPC in the formation of new vascular networks in CNV. A previous study³² investigated the effect of three-monthly injections of anti-VEGF in 23 nvAMD patients and found that the anti-VEGF treatment is associated with a partial normalization of the systemic EPC profile. In fact, at variance with our study, in AMD patients, the authors detected a lower number of EPCs than that observed in the 20 control subjects and hypothesized that bone marrow–derived endothelial cells may significantly contribute to the CNV process in patients with AMD. In contrast with Scotti et al.’s work,³² we documented, at 120 days after the anti-VEGF treatment, a significant reduction of EPC, but not of CPC or CEC, thus supporting the concept that a reduction of EPC may contribute, at least in part, to reduce CNV. Studies conducted on animal models evidenced that treatment agents preventing homing and/or adhesion of progenitor cells to a damaged choroid could reduce CNV,^11,36 and in humans, anti-VEGF agents resulted effective in the treatment of nvAMD and other ocular diseases such as diabetic retinopathy and neovascular glaucoma.³⁷ The discrepancy between our results and Scotti et al.’s study,³² which documented that injections of ranibizumab is associated with the increase of circulating EPC up to the level of the control subjects, may be explained, at least in part, by the different characteristics of the enrolled patients. In fact, Scottie et al.’s patients³² had a very benign risk factor profile, that is, lower prevalence of cardiovascular risk factors (absence of diabetes and smoking, excellent BMI, normal or well-controlled blood pressure, and moderate use of cardiovascular medications), whereas our population had a high prevalence of cardiovascular risk factors, known to significantly reduce EPC.

EPC are rare in circulation and, in addition to the EPC identification methodology, there are other factors affecting our results. For instance, it is known that the expression of the EPC results as cell counts per unit of volume may be not appropriate when dealing with rare events, because it leads to a magnification of the measurement error. Rather, EPC counts should be always reported as the number of cells per total number of cytometric events. This will eliminate any bias related to variation in body fluids and total blood cell counts.³⁸ However, in Scotti et al.’s study,³² the first ranibizumab injection did not affect the EPC number measured on Days 4 and 30, and on Day 120, there was only a tendency toward an increase in EPC. In addition, the evaluation through the functional assay as CFU-Hill cells failed to demonstrate a significant effect of anti-VEGF agent on EPCs also after three injection of ranibizumab. As in our study we measured progenitor and endothelial cells in the systemic circulation, we cannot demonstrate a direct mechanistic relation between anti-VEGF treatment and reduction of EPC. Although some clinical studies have shown that intravitreal ranibizumab does not exert major systemic effects,^17,39–41 three injections of ranibizumab exerted a beneficial effect on the CNV and also on EPC number, suggesting that the blockage of VEGF action at the CNV site is able to reduce the EPC recruitment.¹¹ Ultimately, clinical studies have demonstrated that a low number of circulating EPC was associated with increased cardiovascular risk^42–45 and systemic VEGF inhibition disrupts endothelial homeostasis and accelerates the atherogenesis, suggesting that these events contribute to the clinical cardiovascular adverse events of VEGF-inhibiting therapies.⁴⁶

Our results, showing a systemic effect (lowering of the EPC level) of the anti-VEGF drug, suggest a possible effect of these therapies on cardiovascular risk; however, there is no clear evidence that treatment with intravitreal anti-VEGF in the patients with nvAMD increases cardiovascular risk predictors.^47–50

The main limitation of this study is the small number of examined patients. However, the sample size of our study is similar to those already present in the literature. The second limitation is the lack of multiple determinations of EPC, CPC, and CEC at different timing points in treated nvAMD patients, but a previous study has documented no significant changes after the first anti-VEGF injection.³² Third, we do not assess circulating levels of VEGF and stromal-derived-factor-1 (SDF-1), two factors known to induce the EPC mobilization from bone marrow. Deficiencies in these factors may lead, at least in part, the down-regulation of EPC number and activity.

In conclusion, our findings indicate that CEC number is associated with the CNV in AMD and that anti-VEGF treatment is associated with an EPC reduction. These results support the hypothesis of an involvement of endothelial dysregulation in AMD and suggest that an anti-VEGF therapy may affect the responsiveness of circulating EPC to the CNV process in AMD.

Supplemental Material

Table_4 – Supplemental material for Circulating endothelial and progenitor cells in age-related macular degeneration

Supplemental material, Table_4 for Circulating endothelial and progenitor cells in age-related macular degeneration by Dario Pasquale Mucciolo, Rossella Marcucci, Andrea Sodi, Francesca Cesari, Vittoria Murro, Angela Rogolino, Stanislao Rizzo, Betti Giusti, Gianni Virgili, Domenico Prisco and Anna Maria Gori in European Journal of Ophthalmology

Footnotes

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Supplemental material

Supplemental material for this article is available online.

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