Argonaute-2 promotes miR-18a entry in human brain endothelial cells

Abstract

Background: Cerebral arteriovenous malformation (AVM) is a vascular disease exhibiting abnormal blood vessel morphology and function. miR-18a ameliorates the abnormal characteristics of AVM-derived brain endothelial cells (AVM-BEC) without the use of transfection reagents. Hence, our aim was to identify the mechanisms by which miR-18a is internalized by AVM-BEC. Since AVM-BEC overexpress RNA-binding protein Argonaute-2 (Ago-2) we explored the clinical potential of Ago-2 as a systemic miRNA carrier.

Methods and results: Primary cultures of AVM-BEC were isolated from surgical specimens and tested for endogenous miR-18a levels using qPCR. Conditioned media (CM) was derived from AVM-BEC cultures (AVM-BEC-CM). AVM-BEC-CM significantly enhanced miR-18a internalization. Ago-2 was detected using western blotting and immunostaining techniques. Ago-2 was highly expressed in AVM-BEC; and siAgo-2 decreased miR-18a entry into brain-derived endothelial cells. Only brain-derived endothelial cells were responsive to the Ago-2/miR-18a complex and not other cell types tested. Secreted products (eg, thrombospondin-1 [TSP-1]) were tested using ELISA. Brain endothelial cells treated with the Ago-2/miR-18a complex in vitro increased TSP-1 secretion. In the in vivo angiogenesis glioma model, animals were treated with miR-18a in combination with Ago-2. Plasma was obtained and tested for TSP-1 and vascular endothelial growth factor (VEGF)-A. In this angiogenesis model, the Ago-2/miR-18a complex caused a significant increase in TSP-1 and decrease in VEGF-A secretion in the plasma.

Conclusions: Ago-2 facilitates miR-18a entry into brain endothelial cells in vitro and in vivo. This study highlights the clinical potential of Ago-2 as a miRNA delivery platform for the treatment of brain vascular diseases.

Keywords: Argonaute‐2; angiogenesis; arteriovenous malformation; brain endothelial cells; miR‐18a.

Figure 1.

AVM‐BEC‐conditioned media (AVM‐BEC‐CM) potentiates miR‐18a internalization. A, AVM‐BEC and control BEC were analyzed for intracellular miR‐18a levels using qPCR. Control BEC were used as baseline (n=3; ***P<0.001; paired t test). B, miR‐18a (40 nmol/L) in combination with AVM‐BEC‐CM (black bars) or fresh culture media (white bars) was added to AVM‐BEC and tested for intracellular miR‐18a after 5, 10, 30 minutes (n=4), 1, 2, 8 and 24 hours (n=3). AVM‐BEC‐CM enhanced miR‐18a entry up to 30 minutes (*P<0.05, **P<0.01, ***P<0.001; paired t test). C, Control BEC were treated with miR‐18a (40 nmol/L) in combination with AVM‐BEC‐CM (black bar), control BEC‐CM (gray bar) or fresh media (white bar). Intracellular miR‐18a was analyzed (qPCR) after 30 minutes incubation; AVM‐BEC‐CM potentiated miR‐18a internalization by control BEC (n=3; *P<0.05; paired t test). D, Control BEC were treated with miR‐18a (40 nmol/L) and in the presence of serial diluted AVM‐BEC‐CM (diagonal line bars), demonstrating that progressively diluted AVM‐BEC‐CM loses its ability to enhance miR‐18a internalization (n=3, *P<0.05, **P<0.01; paired t test). Dotted line represents miR‐18a uptake by control BEC in the presence of fresh media. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction.

Figure 2.

Ago‐2 is highly expressed by AVM‐BEC. A, Basal expression of RNA‐binding proteins (NPM, nucleophosmin‐1; NCL, nucleolin; Ago‐2, argonaute‐2) in AVM‐BEC and control BEC were analyzed by qPCR. Increased levels of NCL and Ago‐2 in AVM‐BEC as compared to control BEC were detected (n=3; *P<0.05; **P<0.01; paired t test). A representative western blot is depicted below showing Ago‐2 increased protein levels in AVM‐BEC compared to control BEC. B, AVM‐BEC were treated with siAgo (30 to 75 nmol/L), scrambled siAgo (50 to 75 nmol/L) and lipofectamine (2 μg/mL) and Ago‐2 protein levels were analyzed by Western blotting. siAgo‐2 (75 nmol/L) decreased ≈50% of intracellular Ago‐2 protein content (n=3; *P<0.05; paired t test). A representative image depicting the effects of siAgo‐2 (75 nmol/L) alone and in the presence of lipofectamine (2 μg/mL) is shown below. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells.

Figure 3.

Ago‐2 silencing compromises miR‐18a entry. A, Intracellular miR‐18a detection in AVM‐BEC, control BEC, tumor‐derived endothelial cells (TuBEC), human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells (HMEC) and astrocytes treated with miR‐18a (40 nmol/L) in the presence of siAgo‐2‐AVM‐BEC‐CM (dotted bars) or AVM‐BEC‐CM (black bars). Ago‐2 silencing decreased miR‐18 entry in AVM‐BEC, control BEC, and TuBEC (n=3; *P<0.05; paired t test). B, Intracellular detection of miR‐18a in control BEC (qPCR) after treating cells for 30 minutes with different concentrations of Ago‐2 (0.01 to 4 nmol/L) in combination with miR‐18a (40 nmol/L) showed that higher concentrations of Ago‐2 (up to 0.4 nmol/L) increased miR‐18a detection (n=3). Dotted line represents miR‐18a uptake by control BEC in the presence of AVM‐BEC‐CM. C, Analysis of intracellular miR‐18a (qPCR) showed that miR‐18a (40 nmol/L) in combination with Ago‐2 (0.4 nmol/L) (for 5, 30, 120 and 1440 minutes) was more resistant to degradation than miR‐18a alone; maximum effect was observed at 120 minutes (n=3; **P<0.01; repeated measures 2‐way ANOVA). D, AVM‐BEC and control BEC were exposed to miR‐18a in combination with siAgo‐2‐AVM‐BEC‐CM or AVM‐BEC‐CM. Ago‐2 staining (red) showed that cells exposed to AVM‐BEC‐CM increased Ago‐2 detection in control BEC when treated with miR‐18a (40 nmol/L) (n=3, **P<0.01). Nuclear staining is shown in blue. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). For experiments using cell lines, (n) is represented by cell cultures obtained from different passages. ANOVA indicates analysis of variance; AVM, arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction.

Figure 4.

Facilitated transport is involved in miR‐18a delivery. A, AVM‐BEC and control BEC were treated with AVM‐BEC‐CM plus miR‐18a (40 nmol/L) at 4°C and 37°C for 30 minutes. Intracellular miR‐18a was measured using qPCR as described previously, showing that at 4°C miRNA entry was only minimally compromised (n=3). B, The distribution of Ago‐2 (red) was identified using immunocytochemistry. At 4°C untreated AVM‐BEC expressed high levels of intracellular Ago‐2 (i) compared to untreated control BEC (ii). When control BEC were treated with AVM‐BEC‐CM plus miR‐18a at 4°C, Ago‐2 staining was apparent and associated with the cell membrane (iii; white arrows) (n=3). Blue staining denotes nuclear staining. C, The formation of a ribonucleoprotein complex between Ago‐2 and miR‐18a was determined by immunoprecipitation and immunoblotting (left panel) and qPCR (right panel). Ago‐2 was detected only in the 2 fractions in contact with anti‐Ago‐2, as expected. Only the fraction with both Ago‐2 and miR‐18, but not miR‐18a alone, led to the detection of miR‐18a by qPCR. Rabbit IgG served as the isotypic control. D, GW4869 (10 to 50 μmol/L), a specific inhibitor of N‐Smase‐2 (neutral sphingomyelinase‐2) did not interfere with miR‐18a uptake in AVM‐BEC. Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction.

Figure 5.

Ago‐2 silencing decreases miR‐18a‐induced TSP‐1 secretion. A, AVM‐BEC were treated with siAgo‐2 (75 nmol/L) followed by miR‐18a treatment (40 nmol/L) and cell supernatants tested for TSP‐1 (n=4; *P<0.05; paired t test). B) Control BEC were treated with varying concentrations of the miR‐18a inhibitory sequence, antagomir (40 to 120 nmol/L). Antagomir treatment (80 nmol/L) significantly decreased TSP‐1 levels (n=4; **P<0.01; paired t test). Data are presented as mean±standard error of the mean (SEM). Each human specimen‐derived cell culture represents a unit of analysis (n). AVM indicates arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; TSP‐1, thrombospondin‐1.

Figure 6.

Co‐treatment of miR‐18a and Ago‐2 in vivo “normalizes” TSP‐1 and VEGF‐A plasma levels. A, Athymic nude mice were implanted with glioma cells intracranially. After 3 days, animals were treated intravenously with vehicle, miR‐18a plus Ago‐2, miR‐18a alone or Ago‐2 alone every 48 hours for 3 cycles. Subsequently, plasma was tested for TSP‐1 (A) and VEGF‐A (B). miR‐18a and Ago‐2 combination treatment caused the most significant increase of TSP‐1 levels (n=5; *P<0.05; ***P<0.001; paired 1‐way ANOVA followed by Dunnett's Multiple Comparison Test), and reduction of VEGF‐A levels (n=5; *P<0.05; paired 1‐way ANOVA followed by Dunnett's Multiple Comparison Test). Control plasma (healthy) was obtained from normal athymic mice. C, qPCR analysis of miR‐18a detection showed that tumor cells did not internalize miR‐18a in the presence of AVM‐BEC‐CM (black bars) or Ago‐2 (white bars) (n=3). D, Imaging of intracranial renilla luciferase‐positive tumor cells day 3 after implantation (beginning of treatment) and day 9 (treatment completion). E, Region of interest (ROI) analysis of detected luminescence showed that miR‐18a and Ago‐2 combination and miR‐18a treatment alone led to slower tumor growth as compared to vehicle‐treated animals, albeit not statistically significant (n=5). Data are presented as mean±standard error of the mean (SEM). ANOVA indicates analysis of variance; AVM, arteriovenous malformation; Ago‐2, Argonaute‐2; BEC, brain endothelial cells; CM, conditioned media; qPCR, quantitative real‐time polymerase chain reaction; TSP‐1, thrombospondin‐1; VEGF, vascular endothelial growth factor.