Abnormal Complement Activation and Inflammation in the Pathogenesis of Retinopathy of Prematurity

Abstract

Retinopathy of prematurity (ROP) is a neurovascular complication in preterm babies, leading to severe visual impairment, but the underlying mechanisms are yet unclear. The present study aimed at unraveling the molecular mechanisms underlying the pathogenesis of ROP. A comprehensive screening of candidate genes in preterms with ROP (n = 189) and no-ROP (n = 167) was undertaken to identify variants conferring disease susceptibility. Allele and genotype frequencies, linkage disequilibrium and haplotypes were analyzed to identify the ROP-associated variants. Variants in CFH (p = 2.94 × 10^-7), CFB (p = 1.71 × 10^-5), FBLN5 (p = 9.2 × 10^-4), CETP (p = 2.99 × 10^-5), and CXCR4 (p = 1.32 × 10^-8) genes exhibited significant associations with ROP. Further, a quantitative assessment of 27 candidate proteins and cytokines in the vitreous and tear samples of babies with severe ROP (n = 30) and congenital cataract (n = 30) was undertaken by multiplex bead arrays and further validated by western blotting and zymography. Significant elevation and activation of MMP9 (p = 0.038), CFH (p = 2.24 × 10^-5), C3 (p = 0.05), C4 (p = 0.001), IL-1ra (p = 0.0019), vascular endothelial growth factor (VEGF) (p = 0.0027), and G-CSF (p = 0.0099) proteins were observed in the vitreous of ROP babies suggesting an increased inflammation under hypoxic condition. Along with inflammatory markers, activated macrophage/microglia were also detected in the vitreous of ROP babies that secreted complement component C3, VEGF, IL-1ra, and MMP-9 under hypoxic stress in a cell culture model. Increased expression of the inflammatory markers like the IL-1ra (p = 0.014), MMP2 (p = 0.0085), and MMP-9 (p = 0.03) in the tears of babies at different stages of ROP further demonstrated their potential role in disease progression. Based on these findings, we conclude that increased complement activation in the retina/vitreous in turn activated microglia leading to increased inflammation. A quantitative assessment of inflammatory markers in tears could help in early prediction of ROP progression and facilitate effective management of the disease, thereby preventing visual impairment.

Keywords: abnormal angiogenesis; alternative complement pathway; cytokines; genetics; inflammation; microglia/macrophage; premature birth; retina.

Figure 1

Expression of inflammatory molecules in proliferative retinopathy of prematurity (ROP). (A) Differential levels of complement components and apolipoproteins in the vitreous humor of ROP and controls (B) Western blotting of C3 done under non-reducing condition, showing its expression in patients (lane 1 and 2) and controls (lane 3 and 4). Arrows indicate the C3 fragments. Thin black line inserted within gel showing that all the lanes were run on the same gel but were not continuous (C) Hematoxylin and eosin staining of ROP vitreous. Arrowheads indicate degenerated morphology of macrophages at 40×-magnification (irregular shaped with large vacuole, nucleus with a very prominent nucleolus); Staining of ROP vitreous with CD68 marker. Arrowheads indicate the presence of activated macrophages at 10× magnification. (D) Differential levels of inflammatory cytokines in the vitreous humor of ROP and controls. Error bars in (A) and (D) show SEM, *p < 0.05, **p < 0.005, ***p < 0.0005 (ROP vs. controls).

Figure 2

Hypoxia induced microglia secretes inflammatory and angiogenic molecules. (A) Time lapse imaging of cytosolic calcium in microglial cells in normal and hypoxic conditions. Scale bar: 200 µm (B) Comparison of Ca_max (Maximum Fluo-4 intensity) in microglial cells for control and hypoxic condition [Ca²⁺transients were measured for 50 cells (n = 50) for each cases]. Data are presented as box plot and were analyzed using Wilcoxon rank-sum test. (C) Semi-quantitative PCR was used to evaluate the expression of secreted product (HIF-1α, IL-1β, BAX, C3, VEGF) of hypoxia-induced microglial cells treated with no CoCl₂ (lane 1), 100 µM CoCl₂ (lane 2), and 150 µM CoCl₂ (lane 3) and β-actin as control.

Figure 3

Extracellular matrix (ECM) metalloproteinases and cytokines increases in proliferative retinopathy of prematurity (ROP) in both vitreous and tears samples. (A) Differential levels of ECM proteins and its inhibitors in ROP and control vitreous [*p < 0.05, **p < 0.005, *** p < 0.0005 (ROP vs. controls)]. (B) Zymogram shows activation of MMPs in ROP vitreous (lanes 5, 6, 7, 8) as compared to controls (1, 2, 3, 4). (C) Differential levels of cytokines in ROP (5 µL) and control tears (5 µL). (D) Differential levels of MMPs in ROP (5 µL) and control tear (5 µL) [*p < 0.05, **p < 0.005, ***p < 0.0005 (severe ROP vs. mild ROP + premature controls)]. (E) Zymography showing more activation of MMPs in severe ROP (lanes 6, 7) as compared to mild ROP (lanes 4,5) and controls (5 µL tears; lanes 1, 2, 3). Error bars show SEM.

Figure 4

A role of MMPs in tears as biomarker for retinopathy of prematurity (ROP) progression: differential expression of Zymogram band intensities of (A) MMP9 and (B) MMP2 in extended cohort including severe ROP (n = 10), mild ROP_progressed (n = 6), mild ROP_regressed (n = 7), and no ROP premature controls (n = 9) [*p < 0.05, **p < 0.005, ***p < 0.0005 (ROP vs. controls)], AU, arbitrary unit.

Figure 5

Schematic diagram of the proposed mechanism of complement activation and its potential effect on macrophage-mediated angiogenesis in retinopathy of prematurity. Cleavage of complement components C3 into C3a, C3b and further into iC3b, C3dg and C3cα; C4 into C4a and C4b and C5 into C5a and C5b leading to the activation of complement cascade, which in turn activates macrophage/microglia or vice versa. Activated macrophage/microglia secretes MMPs, cytokines, proangiogenic proteins, and reduced angiogenesis inhibitors that may lead to increased vessel proliferation and extracellular matrix (ECM) degradation in turn promoting angiogenesis. Red bars represent upregulation of the complement components observed in the present study.