The Fatty Acid Transporter CD36 Mediates Uptake, Biodistribution, and Cardioprotection by Small Extracellular Vesicles From HEK293 Cells

Figures

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  FIGURE 1. Physical and biochemical characterisation of HEK2930‐sEVs. Nanoparticle tracking analysis (NTA) shows that (A) the most particle‐rich fraction is eluted after 3.0 mL with size exclusion chromatography (SEC) with a concentration of (242 ± 21) ×1010 particles/ml with (B) a mean size of 118 ± 52 nm and mode size 98 nm ± 44 nm. (C) Fraction 3.0 mL has high levels of the three EVs‐specific markers, with a higher presence for CD63, as shown by DELFIA (Dissociation‐enhanced lanthanide fluorescence immunoassay). Data shown as mean ± SEM (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. HEK293: human embryonic kidney cells 293; sEVs: small extracellular vesicles.

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  FIGURE 2. Dose‐response and time‐response uptake of HEK293‐sEVs in human endothelial cells and cardiomyocytes. (A–C) Human coronary artery and coronary microvascular endothelial cells (HCAEC and HCMEC respectively) and human umbilical vein endothelial cells (HUVEC) were treated for 4 h with increasing doses of HEK293‐sEVs. A linear dose‐response was revealed in all endothelial cells, as a 10‐fold increase of sEVs dose results in a 10‐fold increase of cellular uptake (Nluc Nanoluciferase activity). (D–F) sEVs rapidly internalise endothelial cells ((D) HCAEC, (E) HCMEC, and (F) HUVEC) within the first hour of incubation reaching the highest intracellular localisation after 4 h, corresponding to higher Nluc activity. A reduction in luciferase activity after 24 h is observed in all endothelial cells, reflective of the reduced amount of sEVs intracellularly. (G) Uptake of HEK293‐sEVs in adult rat cardiomyocytes is slow and time‐dependent, and high Nluc activity is recorded only after 24 h of sEVs incubation. Data shown as mean ± SEM (n = 4). *p < 0.05, **p < 0.01, ****p < 0.0001.

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  FIGURE 3. The CD36 fatty acid transporter influences HEK293‐sEVs cellular uptake. Cells were treated with 200 µM of SSO, an irreversible inhibitor for CD36, for 1 h prior to 109 HEK293‐sEVs treatment for 1 h. (A) Inhibition of CD36 resulted in a reduced sEVs uptake in HCMEC. CPZ was used as an inhibitor of clathrin‐mediated endocytosis (CME), as a positive control. Combination of the two inhibitors resulted had an additive effect in the reduction of sEVs uptake. (B) Similarly in adult rat cardiomyocytes, CD36 inhibition led to a reduced Nluc activity intracellularly, indicative of a reduced HEK293‐sEVs internalisation. In this cell type, CME pathway was not involved in sEVs uptake. Data shown as mean ± SEM (n = 4). *p < 005, **p < 0.01, ***p < 0.001, and ****p < 0.0001. HEK293: human embryonic kidney cells 293; sEVs: small extracellular vesicles; Nluc: nanoluciferase; SSO: sulfosuccinimidyl oleate sodium; DMSO: dimethyl sulfoxide; CPZ: chlorpromazine.

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  FIGURE 4. Effects of whole‐body perfusion in HEK293‐sEVs biodistribution. C57BL/6 mice were administered with 1010 HEK293‐sEVs and biodistribution was allowed for 1 h, prior to organ and biofluid collection and Nluc quantification in vitro. (A) Less than 0.005% of the initial administered dose was found in either urine, blood cells, or plasma of mice. (B) Major organs of HEK293‐sEVs uptake were the lungs, liver, and the spleen, where Nluc activity was less than 0.1% of the total initial dose injected. Whole‐body perfusion with saline reduced the Nluc activity recorded in these organs substantially, to levels below 0.05%. Data shown as mean ± SEM (n = 3). ****p < 0.0001. HEK293: human embryonic kidney cells 293; sEVs: small extracellular vesicles; Nluc: nanoluciferase.

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  FIGURE 5. Inhibition of the CD36 transporter reduces HEK293‐sEVs biodistribution in the lungs. 12–14 weeks old C57BL/6 male mice were treated with 40 mg/kg of SSO for 1 h before the administration of 1010 HEK293‐sEVs. Biodistribution of sEVs was allowed for 1 h, after which organs and biofluids were collected for the in vitro quantification of the luciferase activity. (A) Nluc activity was less that 0.005% of the total Nluc activity of the initial dose injected all types of biofluids collected. (B) Major organs of sEVs distribution were the lungs, liver, and spleen, in accordance with previous experiments, and global inhibition of CD36 led to a reduction in sEVs uptake in the lungs (**p < 0.01 vs sEVs treated). Data shown as mean ± SEM (n = 3). ****p < 0.0001. HEK293: human embryonic kidney cells 293; sEVs: small extracellular vesicles; Nluc: nanoluciferase; SSO: sulfosuccinimidyl oleate sodium.

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  FIGURE 6. HEK293‐sEVs protect the heart in acute myocardial infarction. C57BL/6 male mice of 12–14 week old were subjected to acute myocardial ischaemia‐reperfusion injury (M I/R). Ischaemia was induced with the ligation of the left anterior descending artery of the left ventricle for 40 min, following by reperfusion for 120 min. 1010 HEK293‐sEVs were injected intravenously at the onset of reperfusion. (A) Administration of HEK293‐sEVs reduced the infarct size compared to control (36 ± 3 % vs. 58 ± 8% respectively, *p < 0.05, n = 5). As a positive control, an IPC (ischaemia pre‐conditioning) group was included, which reduced the infarct size to similar levels with HEK293‐sEVs (31 ± 4%, **p < 0.01 vs. control). No difference in the %AAR was observed in all groups. (B) Representative scanned images of the excised and stained hearts for each group. Following M I/R, hearts were excised and stained with Evan's blue, for the staining of the live, undamaged, tissue, and TTC (triphenyl tetrazolium) for the staining of the area at risk (AAR, red). The ischaemic (dead) tissue appears as white/pale. Data shown as mean ± SEM (n = 5). *p < 0.05 and **p < 0.01. HEK293: human embryonic kidney cells 293;…

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  FIGURE 7. Inhibition of CD36 reverts the cardioprotective effects of HEK293‐sEVs. Mice were subjected to myocardial ischaemia‐reperfusion injury (M I/R; 40 min of ischaemia followed by 120 min of reperfusion). To observe the effects of the CD36 fatty acid transporter in HEK293‐sEVs cardioprotection, the irreversible inhibitor SSO was administered intraperitoneally at 40 mg/kg 1 h before the injection of HEK293‐sEVs (dose: 1010 particles at the onset of reperfusion). (A) sEVs reduced the infarct size compared to control (49 ± 6 % for HEK293‐sEVs vs. 82 ± 3 % for control, ***p < 0.001) and inhibition of CD36 abrogated that effect (63 ± 6 % for HEK293‐sEVs + SSO vs. 39 ± 6 % for HEK293‐sEVs, *p < 0.05). Administration of the inhibitor alone did not affect the infarct size. (B) Cross sections of the hearts for each group. Hearts were stained to visualise the alive tissue (Evan's blue), and then incubated with TTC (triphenyl tetrazolium) to stain the area at risk (red). Subsequently, the dead tissue was visualised in pale/white colour. Data shown as mean ± SEM (n = 5). *p < 0.05, **p < 0.01, and ***p <0.001. HEK293: human embryonic kidney cells 293; sEVs: small extracellular vesicles; IS:…