Role of Complement Components in Retinopathy of Prematurity

Abstract

Purpose: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disorder affecting eye of premature babies with low gestational age (≤32 weeks) and birth weight (≤1,700g). A study on mouse model of ischemia induced retinopathy suggested a protective role for complement C3a and C5a in neovascularisation while complement is known to mediate angiogenesis in Age-Related Macular Degeneration (AMD). However, the exact involvement of complements in ROP pathogenesis is still unclear. The present study aimed to understand the role of complement components in the pathogenesis of ROP.

Methods: Vitreous humor (50-100μl) was collected with prior informed consent from patients (n=40) with stage IV and V of ROP (classified as per ICROP guidelines) along with infants with congenital cataract as control subjects (n=40) undergoing vitrectomy. Prefractionated protein were subjected to trypsin digestion either in-gel or in solution and the resulting peptides were analysed on a FT LTQ Orbitrap Velos mass spectrophotometer. The obtained mass spectra were searched against the International Protein Index (IPI) database using the Peak studio search engine. Following this strategy, we examined the complement components in ROP and control vitreous. Parallely levels of complement component in vitreous were assessed by multiplex ELISA and further validated by western blotting.

Results: Proteomic analysis of in-gel trypsin digested proteins of selected gel pieces ( 160 kDa, 63 kDa and 45 kDa ) detected 371 spectra in ROP while only 95 spectra in controls for C3 fragments ,and 323 spectra in ROP while only 102 spectra in control for C4 fragments. This result showed elevated levels of complement component C3 and C4 in ROP patients which was also confirmed by a parallel multiplex ELISA and in-solution digested total proteome analysis. In gel pieces analysis of 63 kDa and 45 kDa for activated C3 fragments, 182 spectra were found in ROP while 24 spectra were found in controls. Increased number of spectra for activated C3 in ROP vitreous confirmed to the results from western blotting. Cleavage of C5 into C5b was also confirmed by western blotting in ROP.

Conclusions: The elevated levels of complement factors and its activation indicated abnormal immune activity in ROP patients. Activation of complement pathway might be playing an important role in angiogenesis in ROP patients.

Keywords: 706 retinopathy of prematurity • 609 neovascularization • 555 immunomodulation/immunoregulation