Tissue differences in the exosomal/small extracellular vesicle proteome and their potential as indicators of altered tissue metabolism

Abstract

Exosomes/small extracellular vesicles (sEVs) can serve as multifactorial mediators of cell-to-cell communication through their miRNA and protein cargo. Quantitative proteomic analysis of five cell lines representing metabolically important tissues reveals that each cell type has a unique sEV proteome. While classical sEV markers such as CD9/CD63/CD81 vary markedly in abundance, we identify six sEV markers (ENO1, GPI, HSPA5, YWHAB, CSF1R, and CNTN1) that are similarly abundant in sEVs of all cell types. In addition, each cell type has specific sEV markers. Using fat-specific Dicer-knockout mice with decreased white adipose tissue and increased brown adipose tissue, we show that these cell-type-specific markers can predict the changing origin of the serum sEVs. These results provide a valuable resource for understanding the sEV proteome of the cells and tissues important in metabolic homeostasis, identify unique sEV markers, and demonstrate how these markers can help in predicting the tissue of origin of serum sEVs.

Keywords: exosomes; extracellular vesicles; metabolism; proteomics; tissue communication; tissue markers.

Figure 1.. Experimental design and characterization of sEVs released by each cell type

(A) Diagram representing the experimental model. Differentiated white adipocytes (3T3-L1), differentiated immortalized brown adipocytes (BrAd), differentiated C2C12 myotubes, hepatocytes (AML12), and endothelial cells (SVEC) were cultured in exosome-free medium for 48 h and sEVs were collected by differential centrifugation. Protein was isolated and equal amount from each cell type was subjected to a quantitative proteomics based following TMT labeling. (B) Representative electron microscopy images from sEVs isolated from each cell type. Scale bar, 500 nm in the low-magnification pictures (larger squares, above) and 200 nm in the higher-magnification pictures (smaller squares, below). (C) Number of sEVs released by each cell type normalized by the number of cells in the culture plates from which the vesicles were collected. For 3T3-L1 cells, the value corresponds to the average of 4 different clones. (D) Protein concentration determined by bicinchoninic acid (BCA) of the sEVs released to the culture medium by each cell type and normalized by the number of cells present in the culture plates. Data are expressed as means ± SEMs; n = 4. *p < 0.05 between indicated cell type and the other 4, ***p < 0.001 between the indicated cell type and the other 4.

Figure 2.. Proteomic patterns observed in the isolated sEVs

(A) Principal-component analysis (PCA) of the sEV proteome by each cell type showing 2 distinct clusters: hepatocytes/myotubes and endothelium/white adipocytes/brown adipocytes. This and all of the subsequent figures are based on 3 biological replicates for sEVs of each cell type. (B) Heatmap showing some proteins characteristic of each cluster found in the PCA depicted in (A): 12 proteins were enriched in sEV derived from AML12 hepatocytes and C2C12 myotubes, 6 proteins were enriched in sEV from 3T3-L1 adipocytes and BrAd, and 11 proteins were significantly enriched in sEV from SVEC endothelial cells, 3T3-L1 white adipocytes, and BrAd brown adipocytes. (C) Venn diagram showing the number of unique proteins significantly enriched in the sEVs from each cell type and their combinations. The number in the center represents those proteins present at similar levels in the sEVs from all of the cell types. The total number of unique proteins is 349. FDR < 0.25. (D) Heatmap showing the top-10 proteins significantly enriched in the sEVs from each cell type in specific.

Figure 3.. Glycolytic enzymes are enriched in adipocyte-derived sEVs

(A) The glycolytic pathway scheme showing the enzymes involved at each step, their substrates, and their products. In blue are the enzymes found in the sEVs in our experiments. (B) Bar graphs for the sEV relative abundance in the proteomics study in each of the indicated cell types. Data are expressed as means ± SEMs. *p < 0.05 versus all other cell types (n = 3).

Figure 4.. Tetraspanins are not equally expressed in sEVs from different cell types

Identification of other potential sEV markers. (A) Bar graphs for the sEV abundance of CD9 (left), CD63 (center), and CD81 (right) obtained from the proteomics study (n = 3). (B) Representative immunoelectron microscopic images for CD63 of vesicles isolated from C2C12 (up) and SVEC (down). Scale bar, 100 nm. Arrows indicate CD63⁺ signal. (C) Immunoblotting for CD9 in sEVs (upper blot) and cell bodies (lower blot) from the indicated cell types. (D) Bar graphs for the sEV relative abundance given by the proteomics study in the indicated cell types of heat shock family A member 5 (HSPA5, also known as HSP70 or BiP), 14–3-3 protein beta/alpha (YWHAB), colony-stimulating factor 1 receptor (CSF1R), and contactin-1 (CNTN1). Data are expressed as means ± SEMs. n = 3 for sEV samples and n = 4 for cell samples. *p < 0.05 versus all other cell types.

Figure 5.. Immunoblotting of sEV markers for each cell type

Immunoblotting (above) in sEVs (upper panel) and cell bodies (lower panel) and bar graphs from proteomics study (below) from the indicated cell types for adiponectin (ADIPOQ) (A), integrin b1 (ITGB1) (B), secreted protein acidic and cysteine-rich (SPARC) (C), heat shock protein 90AA1 (HSP90) (D), and periostin (POSTN) (E). Data are expressed as means ± SEMs. *p < 0.05 versus all other cell types (n = 3 for sEV and n = 4 for cell samples).

Figure 6.. Changes in serum sEV protein profile in AdicerKO mice

(A) Diagram representing the experimental model. Serum from random-fed control (ADIPOQ-Cre⁻ Dicer^fl/fl) and adipose tissue-specific Dicer knockout mice (AdicerKO, ADIPOQCre⁺ Dicer^fl/fl) was collected, and sEVs were isolated. Equal amounts of protein of each were then subjected to quantitative proteomics using the TMT-labeling technique. (B) Heatmap showing the 86 proteins significantly regulated (FDR < 0.05) in the sEVs from AdicerKO versus control. (C) Top 10 upregulated biological processes determined by Gene Ontology (GO) from the 40 sEV proteins upregulated in the serum from AdicerKO compared to control mice (FDR < 0.01). (D) Relative abundance by the proteomics assay for selected upregulated proteins in AdicerKO compared to control mice (n = 6). (E) Top 10 downregulated biological processes from the 46 downregulated sEV proteins determined by GO in the serum from AdicerKO mice compared to control mice (FDR < 0.01). (F) Relative abundance measured by the proteomics assay of selected downregulated proteins in AdicerKO compared to control mice (n = 6). Data are expressed as means ± SEMs. *p < 0.05 AdicerKO-versus control-derived sEVs.

Figure 7.. High-molecular-weight ADIPOQ is downregulated in sEV from AdicerKO mice

(A) Venn diagram representing the intersection of the 86 differentially regulated sEV proteins from AdicerKO vs control (large circle) with the 12 white adipocyte 3T3-L1 derived (left circle) and 17 BrAd derived (right circle) from our in vitro comparative proteomics. (B) Heatmap showing the protein abundance in sEVs from AdicerKO and control sera for the 5 sEV proteins predicted to be derived from adipocytes. (C) Western blot for EFEMP1/fibulin-3 (up) and PGAM1 (down) in brown adipose tissue (BAT) (n = 4 for control and 5 for AdicerKO) and subcutaneous inguinal white adipose tissue (sWAT) (n = 5 for control and AdicerKO). (D) Protein quantification from the blots depicted in (C) (n = 4 for control and n = 5 for AdicerKO for BAT and n = 5 for control and AdicerKO for sWAT). (E) Representative immunoelectron microscopic images for ADIPOQ in serum sEVs. Scale bar, 200 nm in both pictures. (F) ADIPOQ levels detected by ELISA in serum, sEV-free serum, and sEVs, from AdicerKO and control mice (n = 5). (G) ADIPOQ western blot from serum, sEV-free serum, and isolated sEV from control (Ctl) and AdicerKO (KO) mice. HMW indicates high molecular weight ADIPOQ, while MMW refers to medium molecular weight ADIPOQ. Samples were pooled: wild type (WT) n = 2 and AdicerKO n = 3 (3 animals per sample). (H) sEVs were isolated and left untreated (first lane) or treated with either 100 µg/mL recombinant proteinase K or 0.5% v/v Triton X-100, or both (representative of 2 independent experiments). SDS gels for experiments shown in (G) and (H) were performed under non-reducing conditions. Ponceau staining was used to normalize the tissue abundance of EFEMP1/fibulin-3 and PGAM1 (Figure S7F). Data are expressed as means ± SEMs. *p < 0.05 versus all other cell types.