Cardioprotection mediated by exosomes is impaired in the setting of type II diabetes but can be rescued by the use of non-diabetic exosomes in vitro

Abstract

Many patients with ischaemic heart disease also have diabetes. As myocardial infarction is a major cause of mortality and morbidity in these patients, treatments that increase cell survival in response to ischaemia and reperfusion are needed. Exosomes-nano-sized, lipid vesicles released from cells-can protect the hearts of non-diabetic rats. We previously showed that exosomal HSP70 activates a cardioprotective signalling pathway in cardiomyocytes culminating in ERK1/2 and HSP27 phosphorylation. Here, we investigated whether the exosomal cardioprotective pathway remains intact in the setting of type II diabetes. Exosomes were isolated by differential centrifugation from non-diabetic and type II diabetic patients, from non-diabetic and Goto Kakizaki type II diabetic rats, and from normoglycaemic and hyperglycaemic endothelial cells. Exosome size and number were not significantly altered by diabetes. CD81 and HSP70 exosome markers were increased in diabetic rat exosomes. However, exosomes from diabetic rats no longer activated the ERK1/2 and HSP27 cardioprotective pathway and were no longer protective in a primary rat cardiomyocytes model of hypoxia and reoxygenation injury. Hyperglycaemic culture conditions were sufficient to impair protection by endothelial exosomes. Importantly, however, exosomes from non-diabetic rats retained the ability to protect cardiomyocytes from diabetic rats. Exosomes from diabetic plasma have lost the ability to protect cardiomyocytes, but protection can be restored with exosomes from non-diabetic plasma. These results support the concept that exosomes may be used to protect cardiomyocytes against ischaemia and reperfusion injury, even in the setting of type II diabetes.

Keywords: Exosomes; cardioprotection; diabetes; ischaemia; reperfusion.

Figure 1

The concentration and modal size of exosome preparations as determined by Nanoparticle tracking analysis. The concentration (A) and size (B and C) of exosomes isolated from non‐diabetic rats and GK diabetic rats. N = 5 per group. The concentration (D) and size (E and F) of exosomes isolated from patients with or without type 2 diabetes. N = 3 per group. The effect of hyperglycaemic conditions (20 mM Glucose), or iso‐osmotic control conditions (20 mM Mannitol), compared to normoglycaemic conditions (4.5 mM Glucose), on the concentration of exosomes isolated from HUVECs (G), and their average size (H and I). N = 8 per group. Statistical comparison was by t‐test (A and D), or repeated measures anova performed on the raw concentration values (G).

Figure 2

Transmission electron microscopy (TEM) of exosomes isolated from the blood of rats (A and B), human patients (C and D) or from cultured HUVECs under the cultured conditions indicated (E–G). Bar = 100 nm. (H–J) Size distribution diameters from TEM images of vesicles from rats (H) (N = 399 and 505), humans (I) (N = 297 and 284) and HUVECs (J) (N = 59, 294 and 131). No significant difference was found when distributions were compared by Mann–Whitney U‐test or Kruskal–Wallis test for 2 or 3 groups, respectively.

Figure 3

Content of exosome marker proteins CD81 and HSP70 in exosomes isolated from rats (A and B), or HUVECs (C). Markers were measured using an ELISA‐based ‘DELFIA’ assay (see methods). N = 5 (A and B) or 4 (C). *P < 0.05. (D) Quantitative immunostaining of levels of the cmHSP70.1 epitope of HSP70 determined by a flow cytometry‐based assay of exosomes isolated from non‐diabetic rats and GK rats (N = 4). Statistical comparison was by t‐test (A, B, D), or anova (C).

Figure 4

Exosomes from diabetic rats or humans or from endothelial cells cultured under hyperglycaemic conditions were unable to protect primary adult cardiomyocytes from death after hypoxia and reoxygenation. Cardiomyocytes were exposed to 10⁸/ml exosomes from non‐diabetic or GK rats (A), non‐diabetic or diabetic patients, (B), or 10⁷/ml exosomes from HUVECs cultured in 20 mM glucose (gluc) or 20 mM mannitol (man). (C). The cardioprotective agent, insulin was used as a positive control in each case. Cell death was assessed by propidium iodide staining. N = 5(A), 3(B) or 6 (C) independent experiments. Statistical comparison was by repeated measures anova. *P < 0.05; **P < 0.01; ***P < 0.001; ns = non‐significant in the comparisons indicated.

Figure 5

Altered levels of exosomal HSP70 or its glycation do not appear to explain the loss of cardioprotection. (A) Cardiomyocytes were treated with 1 ng/ml HSP70 that had been incubated 7 days in 20 mM glucose or 20 mM mannitol, prior to exposure to hypoxia and reoxygenation injury. Cell death was assessed by propidium iodide staining. TAK242 is an inhibitor of TLR4 signalling. (B) Western blot analysis of HSP70 treated as indicated, and probed using antibodies against HSP70 or the glycation end‐product, argpyrimidine. N = 4 independent experiments. Results were compared by repeated measures anova. ***P < 0.001; ns = non‐significant.

Figure 6

Phosphorylation of ERK1/2 and of HSP27 was significantly increased in rat cardiomyocytes treated for 5 min. with 10⁸/ml exosomes from non‐diabetic rats, but not with those treated with exosomes from GK rats (N = 4) (A–C). Fold increase was compared by t‐test. *P < 0.05.

Figure 7

Exosomes from non‐diabetic rats protect primary adult cardiomyocytes isolated from GK diabetic rats from death after hypoxia and reoxygenation. Cardiomyocytes were treated with 10⁸/ml exosomes from GK rats. Insulin was used as a positive control. N = 4 independent experiments. Results were compared by repeated measures anova. ***P < 0.001.