Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 1. Atheroprotective shear stress (SS) to endothelial cells (ECs) suppresses microRNA-126 (miR-126) in the cocultured smooth muscle cells (SMCs). A, Schematic diagrams of EC/SMC coculture and flow system. B, Human umbilical vein ECs and human umbilical artery SMCs were seeded on the lower and upper sides of a membrane, respectively (EC/SMC). Controls had SMCs but no ECs on the other side (Ø/SMC). The temporal expression levels of primary (pri–miR-126) and mature (miR-126) forms of miR-126 were determined by quantitative reverse transcription–polymerase chain reaction. *P<0.05 vs Ø/SMC 0-hour static. C, The levels of miR-126 in the 24-hour static or sheared media. *P<0.05 vs static control. D, The levels of miR-126 in SMCs incubated with medium 199 supplemented with 2% fetal bovine serum (control [CL] media) or conditioned medium from static or sheared ECs (24-hour shearing) (EC-CM). *P<0.05 vs CL media. E, miR-126 expression in EC monoculture or SMC coculture for 24 hours. F, miR-126 expression in ECs exposed to laminar SS (LSS) for 24 hours. G, The levels of pri–miR-126 and miR-126 in EC coculture with SMCs under 24–hour static or LSS conditions.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Membrane, Expressing, Reverse Transcription, Polymerase Chain Reaction, Control, Incubation

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 2. Endothelial cells (ECs) and shear stress (SS) regulate expressions of microRNA-126 (miR-126) targets in smooth muscle cells (SMCs). A, The mRNA levels of forkhead box O3 (FOXO3), B-cell lymphoma 2 (BCL2), and insulin receptor substrate 1 (IRS1) in Argonaute2 (Ago2) immunocomplexes in SMCs 48 hours after transfection with microRNA-126 mimics (PRE126) or anti–microRNA-126 inhibitor (AM126). *P<0.05 vs cells transfected with negative controls (negative control mimics [PREC] or negative control inhibitor [AMC]). B, The relative luciferase levels in SMCs cotransfected with PRE126 (left), AM126 (right), or the negative control and luciferase reporter plasmids: empty vector only, wild-type (WT) 3′-untranslated region (3′UTR) of FOXO3, BCL2, or IRS1 and mutants of 3′UTR of FOXO3, BCL2, or IRS1. C, The relative luciferase levels in SMCs transfected with the indicated luciferase reporter plasmids and then incubated with control media or EC-conditioned media (CM). *P<0.05 vs cells transfected with empty vector. #P<0.05 vs cells transfected with plasmids harboring the WT 3′UTRs. D, The protein levels of target genes in SMCs monocultured (Ø/SMC) or cocultured with ECs (EC/SMC) kept under static condition or exposed to laminar shear stress (LSS). Images are representative of triplicate experiments with similar results.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Luciferase, Plasmid Preparation, Incubation, Control

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 3. Endothelial cells (ECs) transfer exogenous microRNA-126 (miR-126) to smooth muscle cells (SMCs). SMCs were incubated with conditioned media (CM) derived from ECs transfected with biotinylated synthetic miR-126 (B-miR-126) or no-miR mock control (Mock) for 3 hours. A, The levels of B-miR-126 in ECs or the EC-CM were determined by pull-down experiments with streptavidin- agarose beads followed by quantitative reverse transcription–polymerase chain reaction, (B) and B-miR-126 in ECs and SMCs was detected with QDot-605–streptavidin conjugates. *P<0.05 vs mock control. C and D, SMCs were cocultured with ECs transfected with B-miR-126 or mock control for 24 hours; the levels of B-miR-126 in SMCs were determined by Northern blot (C) or pull-down experiments with streptavidin-agarose beads (D). *P<0.05 vs mock control.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Incubation, Derivative Assay, Transfection, Control, Reverse Transcription, Polymerase Chain Reaction, Northern Blot

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 4. Functional microRNA-126 (miR-126) transmission from endothelial cells (ECs) to smooth muscle cells (SMCs) is independent of vesicles. A, The levels of miR-126 in SMCs incubated with control (CL) media or the EC-conditioned media (CM) treated with DNase I, RNase, or proteinase K (PK) for 3 hours. *P<0.05 vs CL media. B, The levels of miR-126 in ECs transfected with control small interfering (si)RNA; siCL) or siRNA against Drosha (siDROSHA), Dicer (siDICER1) or Argonaute2 (siEIF2C2) or in SMCs incubated with CL media or EC-CM derived from those transfected ECs. C, The levels of miR-126 in SMCs incubated EC-CM derived from ECs transfected with anti–miR-126 inhibitor anti–miR-126 inhibitor (AM126), miR-126 mimics (PRE126), or the negative controls. *P<0.05 vs siCL or CL media. #P<0.05 vs the negative control inhibitor (AMC) or negative control mimics (PREC) EC-CM. D, The levels of miR-126 in SMCs cocultured with lung ECs isolated from wild-type (WT) or miR-126 knockout mice (KO). *P<0.05 vs Ø/SMC (SMC monocultured). EC-CM was ultracentrifuged to fractionate the components in spin-down pellets (Pellet; which was reconstituted with M199), or the remaining supernatant (Super). The levels of miR-126 in (E) the fractionated EC-CM or in (F) SMCs incubated with total or fractionated EC-CM for 3 hours. G, The transcript levels or (H), protein levels of indicated genes in SMCs incubated with the fractioned EC-CM. Images are representative of triplicates with similar results. *P<0.05 vs CL media.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Functional Assay, Transmission Assay, Incubation, Control, Transfection, Derivative Assay, Negative Control, Isolation, Knock-Out

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 5. Argonaute2 (Ago2) associates with microRNA-126 (miR-126) and facilitates its transmission. A, The levels of miR-126 in endothelial cell–conditioned media (EC-CM) immunocomplexes of Ago2 or Ago1 or control immunoglobin G (IgG). *P<0.05 vs IgG control. B, Smooth muscle cells (SMCs) were cocultured with bovine aortic ECs transfected with a plasmid encoding the eGPF-Ago2. The enhanced green fluorescent protein-Ago2 proteins were visualized in live ECs or SMCs by microscopy. C, The protection of miR-126 with preincubation of bovine serum albumin (BSA) or recombinant human Ago2 protein (rAgo2) for 30 minutes after RNase A digestion for 15 minutes. *P<0.05 vs miR-126 only. D, The enrichment of miR-126 in SMCs treated with miR-126 preincubated with BSA or rAgo2 for 3 hours. *P<0.05 vs miRs only. E, The levels of Ago2 in ECs (left) or EC-CM (right) under static or laminar shear stress (LSS) conditions for 24 hours. F, The levels of miR-126 in Ago2 immunocomplex in EC-CM under static of LSS conditions. Images are representative of triplicates with similar results. Semiquantification results are shown in the lower panels. *P<0.05 vs static.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Transmission Assay, Control, Transfection, Plasmid Preparation, Microscopy, Recombinant, Shear

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 6. Shear stress (SS)–imposed endothelial cells (ECs) modulate smooth muscle cell (SMC) turnover through microRNA-126 (miR-126). SMCs were monocultured or cocultured with (1) ECs kept under static condition or exposed to laminar shear stress (LSS) for 24 hours; or (2) ECs transfected with negative control inhibitor (AMC) or anti–miR-126 inhibitor (AM126). A, Proliferating cell nuclear antigen (PCNA) in the SMCs was detected by immunostaining. B, Protein levels of PCNA, cyclinA, and p21 in SMCs were assayed by Western blot. C and D, SMC proliferation and apoptosis analyzed for propidium iodide–stained DNA content and Annexin V–stained membrane flip, respectively. *P<0.05 vs Ø/SMC (SMC monocultured). #P<0.05 vs Static EC-SMC or AMC EC-SMC.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Shear, Transfection, Negative Control, Immunostaining, Western Blot, Staining, Membrane

Journal: Circulation Research

Article Title: Regulation of Vascular Smooth Muscle Cell Turnover by Endothelial Cell–Secreted MicroRNA-126

doi: 10.1161/circresaha.113.280883

Figure Lengend Snippet: Figure 7. Depletion of microRNA-126 (miR-126) in mice inhibits smooth muscle cell (SMC) proliferation and neointimal formation in arteries after cessation of blood flow. A, The miR-126 expression levels in mouse carotid arteries with or without ligation. Vascular endothelial cells (ECs) were denuded before RNA isolation. *P<0.05 vs unligated control. B, In situ hybridization of miR-126 in ligated or unligated carotid arteries from miR-126 wild-type (WT) mice. Yellow arrows, elastic lamina (EL). C, Representative images of modified Verhoeff-Van Gieson–stained ligated or unligated carotid arteries from WT or miR-126 knockout (KO) mice. Arrows indicate the thickened neointima. D and E, Representative images of immunofluorescent-stained proliferating (proliferating cell nuclear antigen [PCNA]) SMC (smooth muscle-α actin) in ligated or unligated carotid arteries from WT and KO mice. F, Representative images of modified Verhoeff-Van Gieson–stained ligated carotid arteries from WT or KO mice treated with control (CL) media or EC-conditioned media (EC-CM); or with local delivery of CL oligo or miR-126 mimics (PRE126). Arrows indicate thickened neointima. G, In situ hybridization of miR-126 in ligated carotid arteries from KO mice with treatment of EC-CM, miR-126, or the respective controls.

Article Snippet: Human umbilical vein ECs (HUVECs) were cultured in medium 199 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 10% Endothelial Growth Medium (Cell Applications).

Techniques: Expressing, Ligation, Isolation, Control, In Situ Hybridization, Modification, Staining, Knock-Out