Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

Abstract

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

Keywords: Cardiomyocytes; Exosomes; Myocardial angiogenesis; Type 2 diabetes; miR-320.

Fig. 1

Proliferation and migration of mouse cardiac endothelial cells (MCECs) are inhibited when co-cultured with cardiomyocytes isolated from GK rats, whereas they are promoted when co-cultured with Wistar (WT) rat cardiomyocytes. (A) A scheme of cell co-culture system in which cardiomyocytes were cultured in the lower chamber of a 12-well plate pre-coated with mouse laminin (10 μg/ml) and MCECs were cultured in the upper chamber of a 12-well insert. (B) GK myocytes inhibited MCEC proliferation, which was promoted by WT myocytes. (C) A scheme of the transwell experiment to evaluate endothelial cell migration. Representative endothelial cells which were trans-welled when co-cultured with WT-cardiomyocytes or GK cardiomyocytes. The quantitative results of endothelial cells migrated are shown in (D). (E/F) Cardiomyocyte-mediated regulatory effects on EC migration were negated upon addition of GW4869. n = 4 wells for each group, and similar results were observed in three additional, independent experiments; *p < 0.05 vs. no cardiomyocyes (CMs).

Fig. 2

Characterization of exosomes released from WT- and GK-cardiomyocytes. (A) The activity of acetycholinesterase (AChE) was reduced in GK-exosomes collected from the same amount of cardiomyocytes, compared with controls (n = 4 rats, *p < 0.05). (B) The levels of CD63 and CD81 were similar between GK-exosomes and WT-exosomes (100 μg of exosomal protein was loaded for Western-blotting). (C/D) The exosome size was measured using a Zetasizer Nano instrument (n = 4 independent experiments). (E/F) WT-exosomes were examined under electron microscopy.

Fig. 3

Effects of GK-exosomes on angiogenesis. (A) GK-exosomes significantly inhibited MCEC proliferation in a dose-dependent manner (n = 6 wells, *p < 0.05 vs. WT-exosomes, similar results were observed in three independent experiments). (B/C) GK-exosomes (20 μg/ml) attenuated MCEC migration and (D–F) tube-like formation. Conversely, WT-exosomes(20 μg/ml) exhibited pro-angiogenic effects (B–F). Total capillary tube length (E) and tube branch points (F) were measured by analytical software Image-Pro Plus 5.1. Five independent fields were assessed for each well (n = 3 wells for each group). All values were expressed as means ± SD; *p < 0.05 vs. medium controls. Similar results were observed in three additional, independent experiments.

Fig. 4

Cardiomyocyte-exosomes uptake and deliver miR-320 in endothelial cells. (A) The levels of miR-320 and miR-126 were examined in (A) cardiomyocytes and (B) myocyte-derived exosomes. (C) Red fluorescent dye DiD-labeled WT-exosomes and GK-exosomes were uptaken and distributed in the cytoplasm of MCECs. (D) The miR-320 levels were dramatically increased in MCECs upon exposure to GK-exosomes. U6 RNA was used as an internal control for qRT-PCR of cellular RNA analysis. C. elegans miR-39 was used as spike-in control for qRT-PCR of exosomal RNA analysis. (n = 5, *p < 0.01 vs. WT-exosomes.)

Fig. 5

Cardiomyocyte exosomal miR-320 functionally down-regulates its target genes in recipient endothelial cells. (A) Diagrams of plasmid construction in which mouse Hsp20 3′-UTR or its mutation was inserted downstream of the luciferase cDNA. (B/C) Dual luciferase activity assay in MCECs treated with WT-exosomes or GK exosomes. (D/E) Western-blotting assay revealed that the protein levels of Hsp20, IGF-1, and Ets2 were significantly reduced in GK-exosome-treated MCECs. n = 4 independent experiments; *p < 0.05 vs. exosome-null-treated MCECs.

Fig. 6

Overexpression of miR-320 inhibits MCEC migration and tube formation. (A) Diagrams of recombinant adenoviral vectors (Ad.GFP and Ad.miR-320). (B) MCECs were infected with Ad.GFP or Ad.miR-320, and the miR-320 levels were measured with qRT-PCR. U6 RNA was used as an internal control. (C) The protein levels of Hsp20, IGF-1 and Ets2 were significantly reduced in Ad.miR-320-cells, compared with Ad.GFP-cells. Overexpression of miR-320 greatly inhibited MCEC migration (D) and tube formation (E). *p < 0.05 vs. Ad.GFP, n = 3 independent experiments.

Fig. 7

Transportation of miR-320 from cardiomyocytes into MCECs is blocked by the exosome inhibitor GW4869. (A) Schemes of MCECs co-cultured with: (1) WT-myocytes, (2) GK-myocytes and (3) GW4869-treated GK-myocytes. Twenty-four hours later, MCECs were collected for the determination of miR-320 levels by qRT-PCR. Addition of GW4869 prevented the increase of miR-320 in MCECs co-cultured with GK-myocytes (*p < 0.05 vs. controls as indicated). Similar results were observed in three additional, independent experiments. (B) Schemes of MCECs: (1) cultured in the presence of 5 mM D-glucose, (2) in the presence of 25 mM D-glucose, (3) co-cultured with WT-cardiomyocytes in the presence of 25 mM D-glucose and (4) co-cultured with WT-cardiomyocytes in the presence of 25 mM D-glucose plus GW4869; schemes of WT cardiomyocytes cultured in the presence of 5 mM D-glucose (5) and in the presence of 25 mM D-glucose (6). The levels of miR-320 were reduced in MCECs under high glucose conditions (2), whereas its levels were remarkably increased when co-cultured with high-glucose-treated cardiomyocytes (3). Such an increase of miR-320 levels may have originated from cardiomyocytes, as miR-320 levels were increased in WT cardiomyocytes upon high-glucose treatment (6), and addition of the exosome inhibitor GW4869 blocked the transfer of miR-320 from cardiomyocytes into MCECs (4). (*p < 0.05 vs. controls as indicated). Similar results were observed in three additional, independent experiments.

Fig. 8

Effects of miR-320-reduced exosomes collected form miR-320-off GK cardiomyocytes on angiogenesis. (A) Infection of GK-cardiomyocytes with Ad.miR-320-off dramatically reduced miR-320 levels in cardiomyocytes and their released exosomes. (B) Protein levels of Hsp20 were remarkably increased in Ad.miR-320-off-infected GK myocytes and their released exosomes. n = 4, *p < 0.05 vs. controls indicated. miR-320-off-exosomes (20 μg/ml) significantly promoted (C) MCEC migration and (D) tube-like formation, compared with miR-off control exosomes. Total capillary tube length was measured as described in Fig. 3. All values were expressed as means ± SD; *p < 0.05 vs. medium controls. Similar results were observed in three additional, independent experiments. (E) Proposed scheme for cardiomyocyte-mediated anti-angiogenesis through exosomal miR-320 in diabetic hearts.