Demographic and clinical characteristics of participants
A total of 174 nAMD patients and 43 healthy controls were recruited to this study. Demographic analysis showed no difference between the two groups in gender distribution, history of hypertension and cardiovascular disease, history of diabetes and family history of AMD (Table 1). There was also no difference in smoking status, body mass index (BMI), and psychiatry drug intake between nAMD patients and healthy controls. However, the average age when blood was taken, the number of participants who took vitamin supplements or low-dose aspirin were significantly higher in nAMD group compared with those in controls (Table 1). Since vitamin supplementation is usually advised to AMD patients, the higher number of people taking vitamin supplements in the nAMD group is likely the consequence of the disease, rather than a potential contributor to the disease. Therefore, this was not included in the subsequent analysis of confounders.
Plasma LCN2, MMP9 and MMP9/LCN2 in nAMD patients
The plasma level of LCN2 in nAMD patients was 181.46 ± 73.62 ng/ml, significantly higher than that in healthy controls (152.24 ± 49.55 ng/ml, P = 0.047, independent samples test, Table 2). However, the difference became insignificant after correcting for age or taking low-dose aspirin (p=0.259 and 0.602, respectively, Table 2). A linear regression study uncovered a positive correlation between age and the plasma levels of LCN2 in nAMD patients (r=0.29, p=0.0002, Figure 1A, red line and red dots). No statistically significant correlation (r=0.28, p=0.06. Figure 1A, green line and green dots) was observed in healthy controls. Our results suggest that plasma levels of LCN2 were positively affected by age, particularly in nAMD patients. No correlation was observed between the plasma levels of LCN2 and the number of intravitreal Lucentis injection in nAMD patients (Figure 1B).
There was no difference in the plasma levels of MMP9 and LCN2/MMP9 complex between nAMD patients and healthy controls (Table 2).
We previously observed a higher population of circulating neutrophils in nAMD patients (19). The result was confirmed in this cohort of participants (Figure 2A). To understand if higher numbers of circulating neutrophils contribute to increased LCN2, a correlation analysis was conducted in the participants whose neutrophil data were collected in our study (19). As expected, the population of circulating neutrophils was positively correlated with plasma levels of LCN2 in nAMD patients (r=0.34, p=0.0007, Figure 2B) but not in healthy controls (r=0.057, p=0.77, Figure 2C). Interestingly, no correlation was observed between age and circulating neutrophils in both nAMD patients (Figure 2D) and controls (Figure 2E), suggesting that ageing and circulating neutrophils contribute independently to the increased plasma LCN2 in nAMD patients.
LCN2 in nAMD subgroups
Out of the 174 nAMD participants, 108 had CNV, 32 with RAP, 23 with PCV and 11 were unknown. One-way ANOVA analysis did not reveal any significant difference in the plasma levels of LCN2, MMP9 or MMP9/LCN2 complex among the control, CNV, RAP and PVC groups (p = 0.108, 0.301 and 0.370 respectively, Table 3). Univariate analysis showed that the plasma levels of LCN2 in CNV subgroup were higher than those of control group (p=0.006, Table 3), however, the difference became insignificant after adjusting for age (p=0.064).
LCN2 in nAMD patients with or without macular fibrosis
Out of the 174 nAMD participants, 58 had macular fibrosis and 110 did not have fibrosis and 6 were unknown. One-way ANOVA showed a significant difference in the plasma levels of LCN2 among the three groups (controls, nAMD with fibrosis, nAMD without fibrosis, p=0.048), with the highest levels detected in patients with macular fibrosis (192.77 ± 76.37 ng/ml, Figure 3). Post hoc Tukey test suggested that the difference resided between the groups of controls and nAMD with fibrosis (p=0.037, Figure 3). The mean age of the control group (73.4 ± 8.9) was significantly younger than that of fibrosis group (79 ± 9.2) and non-fibrosis group (77.9 ± 8.1) (p= 0.002, and 0.013 respectively). After adjusting for age, the difference between nAMD with macular fibrosis and controls remains significant (p=0.033, Figure 3).
LCN2 and responsiveness to anti-VEGF therapy or geographic atrophy (GA)
Out of the 174 nAMD patients, 80 (45.98%) patients responded completely, 90 (51.72%) responded partially, and 4 (2.30%) did not respond to the anti-VEGF therapy. Due to the limited number of non-responders in this study, this group was not included in the statistical analysis. There was no significant difference in the plasma levels of LCN2 between controls (154.21±49.74 ng/ml) and partial responders (178.82±72.35 ng/ml) or complete responders (183.90±75.44 ng/ml). In addition, the plasma levels of LCN2 between patients with GA (181.52±73.03 ng/ml, n = 80) and those without GA (178.74±68.69 ng/ml, n = 34) were comparable.