Serum Amyloid A 4 as a Common Marker of Persistent Inflammation in Patients with Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy

Figures & data

Figure 1 Workflow of the AH proteome profile analysis and the validation of DEPs in nAMD and PCV.

Table 1 Significantly Upregulated Proteins for Each Patient Group

Protein ID	Gene Name	Description	Peptides	FC	p-value
Control vs nAMD
P29401	TKT	Transketolase	16	3.3	0.035
P01706	IGLV2-11	Immunoglobulin lambda variable 2–11	4	2.9	0.014
P08571	CD14	Monocyte differentiation antigen CD14	8	2.2	0.000
P07585	DCN	Decorin	10	2.2	0.018
P01599	IGKV1-17	Immunoglobulin kappa variable 1–17	3	2.1	0.024
P02675	FGB	Fibrinogen beta chain	24	2.0	0.027
P02753	RBP4	Retinol-binding protein 4	8	2.0	0.002
P35542	SAA4	Serum amyloid A-4 protein	3	2.0	0.023
P07357	C8A	Complement component C8 alpha chain	15	2.0	0.006
P07360	C8G	Complement component C8 gamma chain	9	2.0	0.004
Control vs PCV
P01706	IGLV2-11	Immunoglobulin lambda variable 2–11	4	3.0	0.006
P01861	IGHG4	Immunoglobulin heavy constant gamma 4	14	2.5	0.036
A0A0B4J1V0	IGHV3-15	Immunoglobulin heavy variable 3–15	2	2.1	0.001
P07360	C8G	Complement component C8 gamma chain	9	1.9	0.018
P08571	CD14	Monocyte differentiation antigen CD14	8	1.9	0.027
Q15223	NECTIN1	Nectin-1	2	1.9	0.045
P07357	C8A	Complement C8 alpha chain	15	1.9	0.041
P35542	SAA4	Serum amyloid A-4 protein	3	1.8	0.044
P00734	F2	Prothrombin	29	1.8	0.008
P00748	F12	Coagulation factor XII	11	1.8	0.026
nAMD vs PCV
Q06033	ITIH3	Inter-alpha-trypsin inhibitor heavy chain H3	6	2.5	0.035

Table 2 Significantly Downregulated Proteins for Each Patient Group

Protein ID	Gene Name	Description	Peptides	FC	p-value
Control vs nAMD
Q08188	TGM3	Protein-glutamine gamma-glutamyltransferase E	12	0.2	0.039
P15924	DSP	Desmoplakin	89	0.3	0.003
Q7Z7M0	MEGF8	Multiple epidermal growth factor-like domains protein 8	3	0.3	0.043
P31944	CASP14	Caspase-14	7	0.3	0.006
P04406	GAPDH	Glyceraldehyde-3-phosphate dehydrogenase	14	0.3	0.041
Q96PG2	MS4A10	Membrane-spanning 4-domains subfamily A member 10	2	0.3	0.033
P32119	PRDX2	Peroxiredoxin-2	9	0.4	0.019
P05089	ARG1	Arginase-1	5	0.4	0.039
Q02413	DSG1	Desmoglein-1	23	0.4	0.010
Q96P63	SERPINB12	Serpin B12	9	0.4	0.024
Control vs PCV
Q96PG2	MS4A10	Membrane-spanning 4-domains subfamily A member 10	2	0.3	0.045
P61626	LYZ	Lysozyme C	8	0.4	0.045
P31944	CASP14	Caspase-14	7	0.4	0.029
Q13404	UBE2V1	Ubiquitin-conjugating enzyme E2 variant 1	2	0.5	0.011
O75223	GGCT	Gamma-glutamylcyclotransferase	4	0.5	0.002
Q08554	DSC1	Desmocollin-1	15	0.6	0.036
Q9NT99	LRRC4B	Leucine-rich repeat-containing protein 4B	3	0.6	0.020
Q8TF72	SHROOM3	Protein Shroom3	2	0.6	0.035
Q13410	BTN1A1	Butyrophilin subfamily 1 member A1	2	0.6	0.001
O95274	LYPD3	Ly6/PLAUR domain-containing protein 3	2	0.6	0.004
nAMD vs PCV
A0M8Q6	IGLC7	Immunoglobulin lambda constant 7	4	0.4	0.001
Q6P387	C16orf46	Uncharacterized protein C16orf46	1	0.5	0.047
Q9NS15	LTBP3	Latent-transforming growth factor beta-binding protein 3	6	0.5	0.013
P81605	DCD	Dermcidin	3	0.6	0.044
Q9Y6R7	FCGBP	IgGFc-binding protein	52	0.6	0.019
Q5VU97	CACHD1	VWFA and cache domain-containing protein 1	4	0.6	0.026

Table 3 Summary of Human Aqueous Humor Samples

	LC-MS/MS Analysis				ELISA Validation
Groups	Control (n=10)	nAMD (n=10)	PCV (n=10)	p value	Control (n=10)	nAMD (n=10)	PCV (n=10)	p value
Patient ID	C1-10	A1-10	P1-10		CV1-10	AV1-10	PV1-10
Gender (M/F)	6/4	7/3	6/4	0.866	16/4	15/5	13/7	0.551
Age (ys)	71.00±8.68	68.80±10.55	68.20±9.86	0.796	71.50±9.40	73.20±9.44	71.05±10.59	0.767

Figure 2 Venn diagram and Volcano plots of DEPs in different comparisons. (A) Up- and downregulated DEPs in different comparisons. (B) Venn diagram identifying 631 co-expressed DEPs among Control, nAMD and PCV, 5 unique DEPs in Control, 17 unique DEPs in nAMD and 24 unique DEPs in PCV. (C–E) Volcano plots showing the distribution of differentially expressed proteins in each comparison. The horizontal axis indicates the fold change (FC), the vertical axis indicates the p value. Red dots are upregulated DEPs, blue dots are downregulated DEPs, and gray dots are proteins without significant differential expression. (|log2(FC)| > 0.58; p value < 0.05).

Figure 3 Heatmap and GO-term analysis of DEPs in different comparisons. Cluster analysis heatmap showing the DEPs in each comparison. Each column represents one biological replicate, each row represents a DEP. DEPs are presented in different colors in the heatmap after normalization. Red represents significantly upregulated proteins, blue represents significantly downregulated proteins, gray represents no quantitative information regarding the proteins. GO classification (Level 2) including BP, MF and CC, which differentiate in blue, red and Orange. The horizontal axis represents the number of DEPs in each functional classification. Top 20 enriched GO terms of significantly DEPs in each comparison are also shown. (A–C) Hierarchical cluster analysis and STRING GO-term enrichment of significantly DEPs between Control and nAMD groups. (D–F) Hierarchical cluster analysis and STRING GO-term enrichment of significantly DEPs between Control and PCV. (G–I) Hierarchical cluster analysis and STRING GO-term enrichment of significantly DEPs between nAMD and PCV. (|log2(FC)| > 0.58; P < 0.05).

Figure 4 KEGG pathway enrichment and PPI network of DEPs in different comparisons. KEGG pathway enrichment analysis are represented with barplot and bubble plot, respectively. For upregulated DEGs, the top enriched pathways are represented in red. For downregulated DEGs, the top enriched pathways are represented in blue. In bubble plot, the horizontal axis is rich factor, the vertical axis is the name of the KEGG pathway. The bubble color scaled the enrichment score. Red represents more significant enrichment. The bubble size scaled the protein number. (A–C) KEGG pathway enrichment and PPI network displays in analysis of the up- and downregulated DEPs in Control vs nAMD group. (D–F) KEGG pathway enrichment and PPI network displays in analysis of the up- and downregulated DEPs in Control vs PCV group. (G and H) KEGG pathway enrichment finds in analysis of the up- and downregulated DEPs in nAMD vs PCV group.

Figure 5 Validation of SAA4 and ROC analysis. (A and B) Comparison of SAA4 protein abundance measured by ELISA (A) with LC-MS/MS-based proteomics (B) measurements. (C and D) ROC curves depicting the classification power of SAA4. Data are presented as means ± SD; * indicates a p value < 0.05, **Indicates a p value < 0.01, ***Indicates a p value < 0.001, ns indicates not significant.

Data Sharing Statement

The data presented in this study are available on request from the corresponding author.