Structural and functional characterization of endothelial microparticles released by cigarette smoke

Abstract

Circulating endothelial microparticles (EMPs) are emerging as biomarkers of chronic obstructive pulmonary disease (COPD) in individuals exposed to cigarette smoke (CS), but their mechanism of release and function remain unknown. We assessed biochemical and functional characteristics of EMPs and circulating microparticles (cMPs) released by CS. CS exposure was sufficient to increase microparticle levels in plasma of humans and mice, and in supernatants of primary human lung microvascular endothelial cells. CS-released EMPs contained predominantly exosomes that were significantly enriched in let-7d, miR-191; miR-126; and miR125a, microRNAs that reciprocally decreased intracellular in CS-exposed endothelium. CS-released EMPs and cMPs were ceramide-rich and required the ceramide-synthesis enzyme acid sphingomyelinase (aSMase) for their release, an enzyme which was found to exhibit significantly higher activity in plasma of COPD patients or of CS-exposed mice. The ex vivo or in vivo engulfment of EMPs or cMPs by peripheral blood monocytes-derived macrophages was associated with significant inhibition of efferocytosis. Our results indicate that CS, via aSMase, releases circulating EMPs with distinct microRNA cargo and that EMPs affect the clearance of apoptotic cells by specialized macrophages. These targetable effects may be important in the pathogenesis of diseases linked to endothelial injury and inflammation in smokers.

Figures

Figure 1. Characterization of microparticles released by CS exposure.

(A) Abundance of circulating endothelial microparticles (EMPs) in human plasma (demographics in Supplementary Table 1). Data represent log-transformed CD31+/CD42b- events. ANOVA (p < 0.05; Bartlett’s *p < 0.0001). (B) Abundance of EMPs released from primary human lung microvascular (HLMVEC) and human pulmonary artery (HPAEC) endothelial cells exposed to CS or ambient air (AC) extract (v:v %; 2 h). Mean + SEM; ANOVA (p < 0.001; Tukey’s *p < 0.05, n = 5). (C–E) Representative images (n = 3) from videos (in Supplementary material) of HLMVEC transduced with plasma membrane-RFP-BacMan and exposed to AC ((C) protruding lamellipodia: arrowhead; insert); or to CS (5%, 1 h, (D) and insets; or 2%, 30 min, (E)). Note EMPs (yellow arrow) released from the plasma membrane at tips of retracting filopodia (arrowhead). (F). Transmission electron microscopy micrograph of heterogeneous human plasma cMPs. (G). Abundance and size distribution of EMPs released from AC- and CS-exposed HLMVEC (5%; 2 h) measured by NanoSight-NS300 (Mean+/−SD; n =  3). CS released mostly EMPs 50 nm–200 nm (mode 129 nm; exosome fraction). t-test *p < 0.05 for sizes 82–92 nm, 107–127 nm, 262–277 nm, 372–387 nm, and 790–820 nm.

Figure 2. CS exposure increases specific miRNAs and ceramide species in mouse cMPs and human EMPs.

(A,B) miRNA expression profiles in MPs. Heatmaps of top 50 individual differentially expressed miRNAs (Increased=red; decreased = blue) detected in cMPs from plasma of AC (pink, n = 3) or CS (3 d; blue, n = 3)-exposed mice (F); or in EMPs from AC (pink; n = 5) or CS (2%, 2 h; blue, n = 5)-exposed HLMVEC isolated from five donors. (C) miRNAs levels measured by RT-PCR intracellular and in EMPs from AC- and CS-exposed HLMVEC (Mean+SEM, n = 4, ANOVA p < 0.05; Sidak’s *p < 0.05). (D) The most abundant ceramide species and total ceramide levels in EMP released from MLEC cells treated with CS (10%; 24 h) or AC control. Levels measured by mass spectrometry, normalized by volume of supernatant used to isolate EMP). Mean ± SEM; Student’s t-test *p < 0.05, n =  3. (E) Relative change in ceramide (vs. AC; dotted line) in intracellular- compared to EMPs compartments in CS (10%; 24 h)-exposed MLEC. Mean+SEM (t-test *p < 0.05, n =  3).

Figure 3. Role of acid sphingomyelinase (aSMase) in the release of CS-induced MPs.

(A) ASM activity in plasma of healthy controls (n = 6) or individuals with COPD (n = 14; Supplementary Table 1). Mean+SEM (t-test *p < 0.05). (B) ASM activity in plasma of mice exposed to CS (3 h) or ambient air. Mean ± SEM; (t-test, *p < 0.01, n =  6). (C) EMPs released by MLEC exposed to CS (10%; 24 h) treated with a pharmacological inhibitor of aSMase (ASMinh; imipramine, 50 μM; 1 h). Mean+SEM (ANOVA p < 0.05; Tukey’s, *p < 0.05, n =  3). (D) EMPs released by MLEC isolated from aSMase deficient mice (ASMko; Smpd1−/−) exposed ex vivo to CS (10%; 24 h); Mean+SEM; ANOVA p = 0.02 (Sidak’s, *p < 0.05, n = 6). (E) Circulating MP (cMPs) released in plasma during CS exposure (3 h) of WT or ASMko mice. Mean + SEM; ANOVA p = 0.01 (Sidak’s *p < 0.05, n = 4–5). (G) cMPs released in plasma following induction of endothelial cell-specific aSMase expression in mice, using tamoxifen (TX, 3 d). Comparison is made between control mice (single transgenics expressing only Tie2::CRE) and aSMase-overexpressing mice (double transgenic, expressing Tie2::CRE-ASMase < flox>; ASM tg). Mean ± SEM (t-test, *p < 0.05, n = 3).

Figure 4. Effect of microparticles on macrophage efferocytosis.

(A) Relative efferocytosis index (fold vs. EMPs-free) of apoptotic Jurkat cells by THP-1 in the presence of MPs (x indicates fold concentration) released by HLMVECs (exposed AC or CS extract, 5%, 16 h, n = 3–4) or by human peripheral blood monocytes (differentiated with 5 ng/mL PMA, 48 h, n = 3). Mean ± SEM; ANOVA (#p < 0.05 vs. untreated; *p < 0.05 vs. AC-treated cells). (B) Relative efferocytosis index (fold vs. EMPs-free) of apoptotic cells in the presence of EMPs (1 x) released by HLMVECs pre-treated with aSMase inhibitor (imipramine; 50μM) prior CS exposure (5%, 16 h, n = 3). Mean ± SEM (ANOVA, *p < 0.05). (C) Relative efferocytosis index (fold vs. cMPs-free) of apoptotic cells by THP-1 in the presence of cMPs from COPD individuals (n = 6). Mean ± SEM. ANOVA p < 0.01 (Sidak’s *p < 0.05). (D–F) Flow cytometry analysis of engulfed Cell Tracker green (CTG)-labeled apoptotic cells by splenocytes (D, left panel) with focus on CTG-gated region with F4/80+/CD11b+ macrophages that engulfed CTG-labeled apoptotic cells ((D) right panels) in spleen homogenates of mice. In situ efferocytosis measured as proportion (%) of F4/80+/CD11b+/CTG+ from total F4/80+/CD11b+ cells (E,F) in C57Bl6/J mice injected with vehicle (PBS, 60 μl) or equal numbers of EMPs from either AC- or CS-exposed MLEC ((E) 10%, 2 h, Mean ± SEM, t-test; *p < 0.05); or injected with cMPs isolated from equal volume of plasma of wild-type or aSMase (Smpd1−/−) KO mice exposed to ambient air (AC) or CS ((F) 3 h; Mean ± SEM, ANOVA p = 0.02; Dunnetts’s post-hoc *p < 0.05 vs. Ctl).