Extracellular Vesicle ITGAM and ITGB2 Mediate Severe Acute Pancreatitis-Related Acute Lung Injury

Abstract

Integrins expressed on extracellular vesicles (EVs) secreted by various cancers are reported to mediate the organotropism of these EVs. Our previous experiment found that pancreatic tissue of mice with severe cases of acute pancreatitis (SAP) overexpresses several integrins and that serum EVs of these mice (SAP-EVs) can mediate acute lung injury (ALI). It is unclear if SAP-EV express integrins that can promote their accumulation in the lung to promote ALI. Here, we report that SAP-EV overexpress several integrins and that preincubation of SAP-EV with the integrin antagonist peptide HYD-1 markedly attenuates their pulmonary inflammation and disrupt the pulmonary microvascular endothelial cell (PMVEC) barrier. Further, we report that injecting SAP mice with EVs engineered to overexpress two of these integrins (ITGAM and ITGB2) can attenuate the pulmonary accumulation of pancreas-derived EVs and similarly decrease pulmonary inflammation and disruption of the endothelial cell barrier. Based on these findings, we propose that pancreatic EVs can mediate ALI in SAP patients and that this injury response could be attenuated by administering EVs that overexpress ITGAM and/or ITGB2, which is worthy of further study due to the lack of effective therapies for SAP-induced ALI.

Keywords: acute lung injury; extracellular vesicles; integrin α M; integrin β 2; pulmonary microvascular endothelial cells; severe acute pancreatitis.

Figure 1

SAP-EV administration disrupts PMVEC connections to promote ALI. (A) Representative lung histology and pathology scores for lung tissue of mice belonging to the sham control (SC) and mild and acute pancreatitis (MAP and SAP) groups (n = 6/group; scale bar = 50 μm, ****p < 0.0001 vs SC, ^####p < 0.0001 vs SAP). (B) Serum EV concentrations for the SC, MAP and SAP groups (n = 3/group; **p < 0.01 vs SC, ****p^< 0.0001 vs SC, ^####p < 0.0001 vs SAP). (C) Representative fluorescence images and statistical analysis of SC and MAP mouse lung tissue after tail-vein injection with SC-, MAP-, and SAP-EV, an equal amount of free DIR, or an equal volume of PBS (n = 3/group; *p < 0.05, **p < 0.01 vs SC-EV, ^#p < 0.05, ^###p < 0.001 vs SAP-EV). (D) Representative lung histology and pathology scores of sham and MAP mice after SC-, MAP-, and SAP-EV injection (n = 6/group, scale bar = 20 μm, ***p < 0.001, ****p < 0.0001 vs SC-EV, ^###p < 0.001 vs SAP-EV). (E) Electron microscopic images of tight junctions between PMVECs in SC mice after the indicated EV injections. Red arrows indicate tight junction connections between PMVECs (scale bar = 2 μm in 8kv, scale bar = 500 nm in 25kv).

Figure 2

SAP-EV abundantly express ITGAM and ITGB2. Extracellular vesicles were extracted from the serum of the three groups mice and analyzed by proteomics. (A) Principal component analysis (PCA) of three groups of EVs (SC-EV, MAP-EV, and SAP-EV) proteomics. (B) Volcano diagram of protein expression differences between SAP-EV and MAP-EV group. (C) GO enrichment analysis of protein expression differences between SAP-EV and MAP-EV group (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Heat map and statistical analyses of integrin protein expression in three groups EVs (n = 3/group; *p < 0.05, ***p < 0.001, ****p < 0.0001 vs SC-EV, ^###p^< 0.001, ^####p < 0.0001 vs SAP-EV). (E) qPCR was used to detect the mRNA levels of integrin-related proteins ILK, ITGA2B, ITGAM, and ITGB2 in the three groups of EVs (n = 6/group; ***p < 0.001, ****p < 0.0001 vs SC-EV, ^#p < 0.05, ^##p < 0.01, ^####p < 0.0001 vs SAP-EV).

Figure 3

HYD-1-inhibition reduces SAP-EV-induced lung injury responses. (A) DIR signal in lungs of healthy mice after tail-vein injection with DIR-labeled SAP-EV, SAP-EV preincubated with HYD-1 (HYD-EV), or SC-EV fractions (n = 3/group; **p < 0.01 vs SC-EV, ^##p < 0.01 vs SAP-EV). (B) Representative histology of EV colocalization with lung tissue microvascular endothelial cells (vWF positive) after EV injection (scale bar = 10 μm). (C) Lung histology and pathology scores of healthy mice after EV injection (n = 10/group; scale bar = 100 μm, ****p < 0.0001 vs SC-EV, ^####p < 0.0001 vs SAP-EV). (D) Lung IL-6 and TNFα expression after EV injection (n = 6/group; ***p < 0.001, ****p < 0.0001 vs SC-EV-lung, ^#p < 0.05, ^##p < 0.01 vs SAP-EV-lung). (E) Lung immunohistology and graphs of VE-cadherin and ZO-1 expression after EV injection (n = 6/group; ***p < 0.001, ****p < 0.0001 vs SC-EV, ^####p < 0.0001 vs SAP-EV).

Figure 4

SAP-EV integrin attenuates PMVEC tight junction proteins expression. (A) Representative histology of SC-EV, SAP-EV, and HYD-EV colocalization with cultured PMVECs (scale bar = 20 μm). (B) Electron microscopy images of PMVECs cell tight junctions (red arrow) after SC-EV, SAP-EV, and HYD-EV incubation (8 kV scale bar = 2 μm, 25 kV scale bar = 500 nm). (C) Western blot analysis and graphs of PMVEC VE-cadherin, ZO-1, and ICAM-1 expression after EV incubation (n = 4/group; **p < 0.01, ****p < 0.0001 vs SC-EV-PMVECs, ^###p < 0.001, ^####p < 0.0001 vs SAP-EV-PMVECs). (D) IL-6 and TNFα expression in PMVEC culture supernatant after EV incubation (n = 6/group; **p < 0.01, ****p < 0.0001 vs SC-EV-PMVECs, ^##p < 0.01, ^####p < 0.0001 vs SAP-EV-PMVECs).

Figure 5

Generation of ITGAM/ITGB2 overexpressing EVs. (A) HEK293T cells transfected with ITGB2, ITGAM, or empty expression vectors to isolate EVs overexpressing ITGB2 (EV^ITGB2) or ITGAM (EV^ITGAM) or negative control EVs (EV^NCV) by ultracentrifugation of culture supernatants. (B) Western blot analysis of ITGB2 and ITGAM expression in transfected cells and their EV isolates. (C) Transmission electron micrographs of EV fractions. (D) Western blot analysis of positive (TSG101, CD9, and Syntenin) and negative (calnexin) EV marker proteins. (E) Size distribution and mean diameter of EV fractions determined by nanoparticle tracking analysis (bar chart: n = 3/group).

Figure 6

EV^ITGB2 and EV^ITGAM treatment blocks lung EV accumulation and inflammation in SAP mice. Pdx1-cre mice were selected to establish SAP model, and then, three groups of engineered EVs (EV^ITGB2, EV^ITGAM, and EV^NCV) labeled with PKH67 fluorescent dye were injected into pdx1-cre-SAP mice by tail vein. (A) Representative images and graph of pancreatic EV accumulation (tdTomato fluorescence) in the lungs of transgenic SAP mice (n = 3/group; ***p < 0.001, ****p < 0.0001 vs SC, ^####p < 0.0001 vs SAP). (B) Representative lung histology and pathology scores of SC, SAP, and EV-treated SAP mice (n = 10; scale bar = 50 μm, ****p < 0.0001 vs SC, ^####p < 0.0001 vs SAP). (C) Transmission electron microscopy images of tight junctions between lung endothelial cells of lung tissue of SC, SAP, and EV-treated SAP mice (8 kV scale bar = 2 μm, 25 kV scale bar = 500 nm). (D) ELISA data for the IL-6 and TNFα concentrations in lung tissue of each group (n = 6/group; ***p < 0.001, ****p < 0.0001 vs SC, ^#p < 0.05, ^###p < 0.001, ^####p < 0.0001 vs SAP).

Figure 7

EV^ITGB2 and EV^ITGAM inhibit SAP-EV-mediated effects to induce PMVEC tight junction disruption and inflammation. Serum EVs of sham control group and SAP group mice (SC-EV and SAP-EV) were labeled with PKH26 fluorescence, and exogenous engineered EVs (EV^ITGB2, EV^ITGAM, and EV^NCV) were labeled with PKH67 fluorescence. Then, two kinds of EVs were used to interfere with primary pulmonary vascular endothelial cells (PMVECs) of mice. (A) Representative images and quantitation of PMVEC uptake of PKH26-stained SC-EV and SAP-EV fractions (red) and PKH67-stained EV^ITGB2, EV^ITGAM, and EV^NCV fractions (green) by DAPI-stained (blue) PMVECs (n = 6/group; ****p < 0.0001 vs SC-EV, ^####p < 0.0001 vs SAP-EV, ^&&&&p < 0.0001 vs SAP-EV+EV^NCV. Tukey’s multiple comparisons test was used to analyze the differences between two groups). (B) ELISA data for IL-6 and TNFα levels in PMVEC culture supernatants after the indicated interventions (n = 6/group; ***p < 0.001, ****p < 0.0001 vs SC-EV, ^#p < 0.05 vs SAP-EV). (C) Representative transmission electron microscopy images of PMVEC junctions after incubation with SC-EV or SAP-EV with or without EV^ITGB2, EV^ITGAM, or EV^NCV (8 kV scale bar = 2 μm, 25 kV scale bar = 500 nm). (D) Western blot analysis and graphs of ICAM-1, VE-cadherin, and ZO-1 in PMVECs after the indicated interventions (n = 6/group; **p < 0.01, ***p < 0.001, ****p < 0.0001 vs SC-EV, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs SAP-EV).