Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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).

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Migration, Cell Culture, Isolation, Co-Culture Assay

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Migration, Software

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.)

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Derivative Assay, Labeling, Control, Quantitative RT-PCR

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Over Expression, Migration, Recombinant, Infection, Quantitative RT-PCR, Control

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Cell Culture, Quantitative RT-PCR

Journal: Journal of molecular and cellular cardiology

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

doi: 10.1016/j.yjmcc.2014.05.001

Figure Lengend Snippet: 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.

Article Snippet: A mouse cardiac endothelial cell line (MCEC) was initially developed in Dr. Weksler’s lab [ 23 ].

Techniques: Infection, Migration, Control