Differential and Altered Spatial Distribution of Complement Expression in Age-Related Macular Degeneration

Abstract

Dysregulation of the alternative complement pathway is a major pathogenic mechanism in age-related macular degeneration. We investigated whether locally synthesized complement components contribute to AMD by profiling complement expression in postmortem eyes with and without AMD.METHODS. AMD severity grade 1 to 4 was determined by analysis of postmortem acquired fundus images and hematoxylin and eosin stained histological sections. TaqMan (donor eyes n = 39) and RNAscope/in situ hybridization (n = 10) were performed to detect complement mRNA. Meso scale discovery assay and Western blot (n = 31) were used to measure complement protein levels. RESULTS.The levels of complement mRNA and protein expression were approximately 15-to 100-fold (P < 0.0001-0.001) higher in macular retinal pigment epithelium (RPE)/choroid tissue than in neural retina, regardless of AMD grade status. Complement mRNA and protein levels were modestly elevated in vitreous and the macular neural retina in eyes with geographic atrophy (GA), but not in eyes with early or intermediate AMD, compared to normal eyes. Alternative and classical pathway complement mRNAs (C3, CFB, CFH, CFI, C1QA) identified by RNAscope were conspicuous in areas of atrophy; in those areas C3 mRNA was observed in a subset of IBA1 + microglia or macrophages. CONCLUSIONS.We verified that RPE/choroid contains most ocular complement; thus RPE/choroid rather than the neural retina or vitreous is likely to be the key site for complement inhibition to treat GA or earlier stage of the disease. Outer retinal local production of complement mRNAs along with evidence of increased complement activation is a feature of GA.

Local factor H production by human choroidal endothelial cells mitigates complement deposition: implications for macular degeneration

“…The RPE has been shown to produce RNA for complement activators and inhibitors locally [20,40,41]. To determine if choroidal endothelial cells also produce CFH locally, we obtained three human donor eyes and separated the choroid from the RPE, extracted RNA, and then generated cDNA to quantify mRNA levels using qPCR.…”

“…However, while the choriocapillaris is a relatively permeable vascular bed due to its numerous fenestrae, the transcellular passage of some glycoproteins of lower molecular weight than FH has been shown to be restricted [17], requiring specific transport via caveolae [18]. In addition to liver-produced complement, certain cell types including the retinal pigment epithelium and microglia in the retina have been shown to be local sources of complement activators and inhibitors [4,19,20]. The contribution of endothelial cells that line the choriocapillaris vessels has yet to be determined.…”

Association of Serum Complement C1q and C3 Level with Age-Related Macular Degeneration in Women

“… 22 Demirs et al demonstrated that in macular RPE/choroid tissue, complement mRNA and protein expression levels are about 15–100 times higher than in normal neuroretinas, and C1qA was one of the components detectable in atrophic areas. 7 …”

“…Secondly, accumulated complement activated products such as C3a, C5a, and membrane attack complex was found to be involved in the formation of drusen and may also be related to the new blood vessels as reported. 7 , 11 The following question is what is the cause of complement activation. A growing body of evidence indicates that AMD pathogenesis involves ocular autoimmunity.…”

“… 6 The increased expression of complement components including C3, CFB, CFH, CFI, and C1qA was found in atrophic regions of AMD patients. 7 Whitcup et al found C3 and C3a and other immune substances accumulated under the retina and caused the activation of complement system and forming of drusen, which was associated with degeneration of retinal pigment epithelial (RPE) cells. 8 The activators and regulatory proteins of complement have also been discovered to be constituents of drusen.…”