Adenosine A 2A Receptor Regulates microRNA‐181b Expression in Aorta: Therapeutic Implications for Large‐Artery Stiffness

Figures

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  Figure 1. Adenosine A2A receptor activation facilitates translin/trax association. A. Western blot for trax (upper band) and translin (middle band) was performed on A7r5 cell lysates harvested after 24 hours of treatment with either vehicle control (0.001% DMSO) or CGS21680 (adenosine A2A receptor–specific agonist). Hsp90 (lower band) was used as a loading control. Band intensity data presented in trax (B) and translin (C) were analyzed by t‐test (n=3/group). D, Following treatment of A7r5 cells with either vehicle control or CGS21680, lysates were harvested and then used for immunoprecipitation with translin antibodies. The pellets were immunoblotted for trax (upper band) and translin (lower band), shown under the IP Translin label. Before adding translin antibodies to the lysate, an aliquot was removed and processed for western blotting for trax, shown under the Total label. The intensities of the immunoprecipitated translin (E) or trax (F) bands were analyzed following normalization to the input translin or trax band intensity for that condition. Data were analyzed by t‐test (n=3/group). *P<0.05 vs control. IP indicates immunoprecipitation.

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  Figure 2. Adenosine A2A receptor activation decreases levels of pre‐miR‐181b and microRNA‐181b via activation of translin/trax RNase. The bar graphs present quantitative PCR data examining the effect of CGS21680 on levels of pre‐miR‐181b (A); microRNA‐181b (B); microRNA‐126a‐3p (C); and let‐7a (D) in total RNA in A7r5 cell lysates harvested after 24 hours of treatment with either vehicle control (0.001% DMSO) or CGS21680 (adenosine A2A receptor–specific agonist). n=3/group. Pre‐ or mature microRNA‐181b, microRNA‐126a‐3p, and let‐7a expression were normalized to RNU6. Data were analyzed by t‐test. *P<0.05, **P<0.01 vs control. Pre‐miR‐181b (E) and microRNA‐181b (F) in total RNA in primary aortic VSMCs from WT‐control mice and translin−/− mice harvested after 24 hours of treatment with either vehicle control (0.001% DMSO) or CGS21680. n=5/group. Pre‐ or mature microRNA‐181b expressions were normalized to RNU6. Data were analyzed by t‐test. **P<0.01, ***P<0.001 vs control. KO indicates knockout; pre‐miR, premature microRNA; VSMCs, vascular smooth muscle cells; TN, translin; and WT, wild‐type.

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  Figure 3. Adenosine A2A receptor activation enhances TGF‐β signaling. A, Western blot was performed on A7r5 cell lysates harvested after 24 hours of treatment with either vehicle control or CGS21680 to detect phospho‐SMAD2/3 (p‐SMAD2/3; upper band) and total SMAD2/3 (t‐SMAD; middle band). Hsp‐90 (lower band) was used as a loading control to normalize the intensity of phospho‐ and total SMAD2/3 bands. B, Band net intensity data were determined by calculating the ratio of p‐SMAD2/3 and t‐SMAD2/3 band intensities and analyzed by t‐test (n=3/group). *P<0.05 vs control.

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  Figure 4. Conditional deletion of translin (Tsn) protects from increased vascular stiffness and decreased microRNA‐181b expression produced by high‐salt water (HSW) stress. A. Weekly pulse wave velocity (PWV) measurements comparing responses of UBC‐CreERT2 X Tsnfl/fl and littermate control (Tsnfl/fl) (n=5) mice treated with HSW. Data were analyzed by 2‐way ANOVA with repeated measures, which showed significant effects of genotype (P<0.0001) and time (P<0.0001), as well as a significant interaction between these variables (P<0.01). B. Tensile testing was performed on the thoracic aorta from UBC‐CreERT2 X Tsnfl/fl and littermate Tsnfl/fl mice treated with HSW (n=3 animals with 4 aortic rings per animal). Data were analyzed by 2‐way ANOVA with repeated measures, which showed significant effects of genotype (P<0.0001) and strain (P<0.0001), as well as a significant interaction between these variables (P<0.0001). Post hoc testing by t‐test at a strain of 2.5 showed P<0.0001. Quantitative polymerase chain reaction data examining pre‐miR‐181b (C) and microRNA‐181b (D) expression in total RNA from aortas of UBC‐CreERT2 X Tsnfl/fl and littermate Tsnfl/fl mice treated with HSW (n=7, 8). Pre‐ or…

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  Figure 5. Adenosine A2A receptor–specific antagonist (SCH58261) protects the aorta from the degradation of pre‐microRNA‐181b/microRNA‐181b and increased vascular stiffness induced by high‐salt water (HSW) stress. A, Pulse wave velocity (PWV) was monitored before the onset of HSW treatment and twice during the 3 weeks of HSW regimen. The graph compares PWV values in mice treated daily with SCH58261 or vehicle control (n=6) during HSW and also with normal drinking water (NW). Data were analyzed by 2‐way ANOVA with repeated measures, which showed significant effects of treatment (P<0.0001) and time (P<0.0001), as well as a significant interaction between these variables (P<0.0001). B, Tensile test was performed on the thoracic aorta from SCH58261‐treated and PBS‐injected mice treated with HSW (n=3 animals with 4 aortic rings per animal). Data were analyzed by 2‐way ANOVA with repeated measures, which showed significant effects of treatment (P<0.0001) and strain (P<0.0001), as well as a significant interaction between these variables (P<0.0001). Post hoc testing by t‐test at a strain of 2.5 showed P<0.0001. Quantitative polymerase chain reaction was performed for pre‐miR‐181b (C), and…

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  Figure 6. Treatment with adenosine A2A receptor–specific antagonist (SCH58261) prevents histological alterations associated with high‐salt water (HSW)‐induced aortic stiffening. A, Left panels show Pre‐miR staining of aortic rings from vehicle control‐ and SCH58216 (SCH)‐injected mice that were treated with HSW for 3 weeks. The intensity of the blue staining, which corresponds to collagen, is shown in the right panel. B, The areas stained only in the media for collagen were quantified and are presented in the bar graph at right, which shows reduced collagen staining in aorta sections from mice that underwent daily SCH58216 treatment. Data were analyzed by t‐test (n=3/group). **P<0.01 vs PBS‐injected. Mice that underwent daily SCH58216 treatment have reduced aortic diameter (C), aortic wall thickness (D), and aortic perimeter (E) compared with control PBS‐injected mice. Data were analyzed by t‐test (n=3/group). *P<0.05, **P<0.01 vs PBS‐injected.

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  Figure 7. Loss of pre‐miR‐181b expression in media of human aorta with aging. A, Representative ×4 staining picture of pre‐miR‐181b in full thickness sections of human aorta of 34‐ and 68‐year‐old men (top) and in higher magnification (×40) views of the middle layer (middle). The bottom panel presents a digitally processed version of these images performed with ImageJ software to display the density of pre‐miR‐181b staining. B, Digitalized slides were analyzed for pre‐miR‐181b stained area using ImageJ software. n=3–4/group. Data were analyzed by t‐test. *P<0.05 vs <40 years group. pre‐miR indicates premature microRNA.

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  Figure 8. Loss of microRNA‐181b expression in VSMCs of human aorta with aging. A, Representative ×4 staining picture of microRNA‐181b in full thickness sections of human aorta from same samples presented in Figure 5 (top). Higher magnification (×40) view of middle layer taken from area indicated on top panel (middle). Digitally processed image showing pre‐miR‐181b staining (bottom). B, Digitalized slides were analyzed for pre‐miR‐181b stained area using ImageJ software. n=3‐4/group. Data were analyzed by t‐test. *P<0.05 vs <40 years group. miR indicates microRNA; and pre‐miR, premature microRNA.

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  Figure 9. Role of adenosine A2A receptor (A2AR) activation in mediating increased aortic stiffness elicited by high‐salt intake or aging. This schematic diagram illustrates key steps in the signaling pathway linking A2AR activation with increased aortic stiffness. According to this model, high‐salt intake or aging increases extracellular levels of adenosine which activate A2ARs on VSMCs. Stimulation of A2ARs triggers dissociation of trax from its C‐terminus enabling trax to bind to translin to form active translin/trax complexes. Enhancing translin/trax RNase activity triggers degradation of its substrate, pre‐miR‐181b, and prevents its conversion into mature microRNA‐181b by Dicer. Decreased silencing of mRNAs targeted by microRNA‐181b leads to increased extracellular levels of TGF‐β, which, in turn, drives aortic stiffening. miR indicates microRNA; pre‐miR, premature microRNA; and VSMCs, vascular smooth muscle cells.