Exosomal myeloperoxidase as a biomarker of deep venous thrombosis

Abstract

Background: Deep vein thrombosis (DVT) often occurs following major orthopedic surgery. In this study, we investigated specific exosomal proteins as potential diagnostic biomarkers of DVT.

Methods: Proteomic analysis of exosomes from four DVT patients and healthy controls (n=4) was performed by mass spectrometry. The model animals were evaluated at 1 inferior vena cava ligation [(IVCL)-1D], 3 (IVCL-3D), and 7 (IVCL-7D) days after IVCL. Endothelial cells in the thrombus segment were examined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and hematoxylin and eosin (HE) staining. Myeloperoxidase (MPO) expression in the damaged vessel was detected by immunofluorescence staining. Exosomes were co-cultured with human umbilical vein endothelial cells (HUVECs) and cell proliferation was estimated using Cell Counting Kit-8 (CCK-8) assays.

Results: A total of 78 differentially expressed proteins (DEPs; 38 downregulated and 40 upregulated) were identified in the DVT group. In the rat DVT model, endothelial cells were damaged continuously after thrombosis, with the most serious injury in the IVCL-3D group, after which signs of endothelial repair were apparent. The IVCL-1D group showed the highest levels of vascular endothelial cell apoptosis and MPO increased sharply in the IVCL-1D and IVCL-3D groups, but had almost disappeared in the IVCL-7D group. In co-culture, plasma exosomes isolated from DVT model rats were efficiently absorbed by HUVECs, with markedly lower HUVECs growth and higher levels of apoptosis in the IVCL-1D and IVCL-3D groups compared with the control group.

Conclusions: Our findings suggest that exosomes may be involved in endothelial cell injury during DVT. The exosomal protein MPO is a potential biomarker of early stage DVT.

Keywords: Exosomes; deep vein thrombosis (DVT); myeloperoxidase; proteomic analysis.

Figure 1

Identification of human plasma exosomes in the experimental group and the control group. (A) Transmission electron micrograph of exosomes (×5,000 nm). (B) Nanoparticle tracking analysis. Exosome diameters ranged from 100 to 150 nm. (C) Higher expression of Alix was observed in the thrombus group compared with the control group. *, P<0.05.

Figure 2

Proteomics analysis of plasma exosomes from patients with DVT and healthy individuals. Heat maps of 24 significantly differentially expressed exosomal proteins obtained from the plasma of participants in the thrombus and control groups. DVT, deep vein thrombosis.

Figure 3

Images of inferior vena cava injury at different time points (1, 3, and 7 days) after ligation. (A) HE staining showing that the continuity and integrity of blood vessels were disrupted following inferior vena cava ligation, and the vessel wall was most severely damaged 3 days after ligation (×40 µm). The black arrows indicate the morphology of the inferior vena cava vessel wall in rats treated with different methods. (B) TUNEL staining showing that venous blood vessel apoptosis was most obvious at 3 days after ligation (×40 µm). The white arrow indicates the apoptotic tissue in the inferior vena cava vessel wall of the rat. IVCL, inferior vena cava ligation; HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 4

The distribution of MPO content in the inferior vena cava over time after inferior vena cava thrombosis. (A) Western blot analysis of MPO expression at different time points after venous thrombosis (1, 3, and 7 days). MPO was higher in the IVCL-1D and IVCL-3D groups, but significantly lower in the IVCL-7D group. Expression of the exosomal markers CD63 and Alix increased after venous thrombosis. (B) Immunofluorescence analysis of the distribution of MPO in the inferior vena cava at different time points after thrombosis. MPO was mainly distributed in the blood vessel wall in the IVCL-1D group and in the emboli in the IVCL-3D group, while it had almost disappeared in the IVCL-7D group (×50 µm). *, P<0.05. DAPI, 4’,6-diamidino-2-phenylindole; MPO, myeloperoxidase.

Figure 5

HUVECs uptake of plasma exosomes containing MPO. (A) Levels of CD63 and Alix in the IVCL-1D, IVCL-3D, and IVCL-7D groups was increased relative to levels of the control group. The content of MPO in the IVCL-1D and IVCL-3D groups increased significantly. (B) Exosomes labeled with green fluorescence were absorbed in large quantities by HUVECs. *, P<0.05. ×200 µm. HUVECs, human umbilical vein endothelial cells; TSG101, tumor susceptibility gene 101; MPO, myeloperoxidase.

Figure 6

Exosomes containing MPO in the plasma of rats from different groups of thrombus models were co-cultured with HUVECs. (A) Relative growth status of HUVECs (IVCL-1D < IVCL-3D < IVCL-7D < control group) observed under a light microscope (×100 µm). (B) CCK-8 assay showing the highest and lowest numbers of HUVECs in the control and IVCL-3D groups, respectively; data are presented as mean ± SD, *, P<0.05. (C) TUNEL staining of apoptosis of HUVECs: IVCL-3D > IVCL-1D > IVCL-7D (×100 µm). The white arrow indicates apoptotic vascular endothelial cells. HUVECs, human umbilical vein endothelial cells; MPO, myeloperoxidase; CCK-8, Cell Counting Kit-8; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.