Exosome-Contained APOH Associated With Antiphospholipid Syndrome

Abstract

Background: Antiphospholipid syndrome (APS) is a systemic autoimmune disease that can lead to thrombosis and/or pregnancy complications. Exosomes, membrane-encapsulated vesicles that are released into the extracellular environment by many types of cells, can carry signals to recipient cells to affect angiogenesis, apoptosis, and inflammation. There is increasing evidence suggesting that exosomes play critical roles in pregnancy. However, the contribution of exosomes to APS is still unknown.

Methods: Peripheral plasma was collected from healthy early pregnancy patients (NC-exos) and early pregnancy patients with APS (APS-exos) for exosome extraction and characterization. The effect of exosomes from different sources on pregnancy outcomes was determined by establishing a mouse pregnancy model. Following the coincubation of exosomes and human umbilical vein endothelial cells (HUVECs), functional tests examined the features of APS-exos. The APS-exos and NC-exos were analyzed by quantitative proteomics of whole protein tandem mass tag (TMT) markers to explore the different compositions and identify key proteins. After incubation with HUVECs, functional tests investigated the characteristics of key exosomal proteins. Western blot analysis was used to identify the key pathways.

Results: In the mouse model, APS-exos caused an APS-like birth outcome. In vitro experiments showed that APS-exos inhibited the migration and tube formation of HUVECs. Quantitative proteomics analysis identified 27 upregulated proteins and 9 downregulated proteins in APS-exos versus NC-exos. We hypothesized that apolipoprotein H (APOH) may be a core protein, and the analysis of clinical samples was consistent with finding from the proteomic TMT analysis. APOH-exos led to APS-like birth outcomes. APOH-exos directly enter HUVECs and may play a role through the phospho-extracellular signal-regulated kinase pathway.

Conclusions: Our study suggests that both APS-exos and APOH-exos impair vascular development and lead to pregnancy complications. APOH-exos may be key actors in the pathogenesis of APS. This study provides new insights into the pathogenesis of APS and potential new targets for therapeutic intervention.

Keywords: MAPK pathway; antiphospholipid syndrome; exosomes; mice model; proteomics analysis.

Figure 1

(A) Transmission electron microscopy (TEM) images of collected exosomes. Scale bars in the images represent 200 nm. (B) Diagram of the particle size distribution obtained by nanoparticle tracking analysis. (C) Confirmation of marker proteins in the exosome (exos) preparations. Western blotting of samples for HSP70, CD81, CD9, CD63, Calnexin and Grp94. (D) The mice were injected with antiphospholipid syndrome (APS)-exos (100 μg/ml/each) or normal control (NC)-exos (100 μg/ml/each) via the caudal vein once a week for 3 continuous weeks. Seven days after the final immunization, the treated female BALB/c mice were mated overnight with male mice. Mating was detected by the appearance of a vaginal plug the following morning. On days 12–14 after mating, all female mice were sacrificed. (E, F) Establishment of the abortion model. Typical uterus and embryos from a mouse treated with APS-exos. (G) The bar shows the percentage of embryo-fetal loss in the different groups. APS-exos increased the abortion rate of mice (NC-exos, n = 7 and APS-exos, n = 5, *P = 0.038 by chi-square test). (H) The scatter plot shows the pregnancy rates in different groups. APS-exos reduced the pregnancy rate. (NC-exos, n=8 and APS-exos, n =10, *P = 0.029 by Mann–Whitney test).

Figure 2

(A) Pathological analysis of hematoxylin and eosin (H&E)-stained placental tissue. H&E staining of placental tissue from the antiphospholipid syndrome-exosomes (APS-exos) and normal control (NC)-exos mouse model analyzed by light microscopy (2× and 20×). Placental blood vessels in mice treated with APS-exos compared with those treated with NC-exos. (B) After incubation with APS-exos or N-exos (50 μg/ml) for 16 h, human umbilical vein endothelial cell (HUVEC) migration was analyzed. *P = 0.029 by Paired t test. (C) After incubation with NC-exos (50 μg/ml) or APS-exos (25 μg/ml, 50 μg/ml, 100 μg/ml) for 2 h and 4 h, angiogenesis of HUVECs was analyzed. *P < 0.05; **P < 0.01; ***P < 0.001 by t test. (D) After incubation with APS-exos/NC-exos (50 μg/ml) for 24h and 48h, HUVEC proliferation was analyzed by the cell counting kit-8 assay. *P = 0.183 by Paired t test. All data shown as mean ± S.D.

Figure 3

Proteomic analysis results. (A) A total of 293 proteins were identified from the two groups. Twenty-seven upregulated proteins and nine downregulated proteins were identified by quantitative proteomics tandem mass tag analysis of antiphospholipid syndrome-exosomes (APS-exos) and normal control (NC)-exos. Mean ± SEM (36 proteins were significantly different, P < 0.05 by t test). (B) The difference in APOH expression between the two groups shown by proteomic analysis (P = 0.000 by t test). (C) Clinical validation showed that the level of APOH was significantly higher in APS-exos than that in NC-exos. “***” means P = 0.000 by t test.

Figure 4

(A) Gene Ontology (GO) annotation was derived from the UniProt-GOA database. GO enrichment analysis of upregulated proteins in the biological process (left), cellular component (mid), and molecular function (right) categories. (B) We enriched and analyzed the entries in GO annotations except for the first three categories (biological process, cellular component and molecular function). (C) The clustering method is based on the p value of Fisher’s exact test obtained from enrichment analysis. The hierarchical clustering method was used to gather relevant functions in different groups and draw them as a heat map. The horizontal of the thermogram represents the enrichment test results of different groups, and the vertical represents the description of differential enrichment related functions (KEGG pathway, protein domain). The different proteins and the color blocks corresponding to the functional description in different groups indicate the degree of enrichment. Red indicates a strong degree of enrichment, while blue indicates a weak enrichment. KEGG pathways with significant enrichment of differentially expressed proteins [antiphospholipid syndrome-exosomes (APS-exos) versus normal control-exos) (above). Cluster analysis heat map based on protein domain enrichment (below).

Figure 5

(A, B) Apolipoprotein H (APOH) lentivirus was transduced into 293T cells to enhance APOH expression. APOH overexpression and control (NC) lentiviral vectors were constructed. The cells and exosomes [N-exosomes (exos) and APOH-exos] were examined by western blotting. (C, D) Establishment of the abortion model is shown in Figure 1D . Typical embryos and uterus from a mouse treated with APOH-exos and NC-exos (50ug/ml/each). (E) APOH-exos increased the abortion rates of mice (NC-exos, n = 7 and APOH-exos, n = 4; *P = 0.012 by Fisher’s exact test). (F) No significant difference in pregnancy rate between the two groups. (NC-exos, n =10 and APS-exos, n =10; *P = 0.319 by Mann–Whitney test). (G) Pathological analysis of hematoxylin and eosin-stained placental tissue from the APOH-exos mouse model by light microscopy (2× and 20×). The placental blood vessels and the vascular thrombus in mice treated with APOH-exos compared with those in mice treated with N-exos.

Figure 6

(A) After incubation with 25 μg/ml APOH-exosome (exos) or 25 μg/ml control (N)-exos for 16 h, human umbilical vein endothelial cell (HUVEC) migration was analyzed. Mean ± SD (*P = 0.014 by Paired t test). (B) After incubation with N-exos (50 μg/ml) or APOH-exos (25 μg/ml, 50 μg/ml, 100 μg/ml) for 2 h and 4 h, HUVEC angiogenesis was analyzed. Mean ± SD (*P < 0.05; **P < 0.01; ***P < 0.001 by t test). (C) After incubation with APOH-exos/N-exos (25 μg/ml) for 24h and 48h, HUVEC proliferation was analyzed by the cell counting kit-8 assay. Mean ± SD (*P = 0.183 by Paired t test). (D) The binding and endocytosis of APOH-exos/N-exos in HUVECs was observed by laser scanning confocal microscopy. (E) After 16 h of incubation of HUVECs with APOH-exos, the levels of Erk 1/2, P-Erk 1/2, p38 and P-p38 were determined by western blotting.