Degradation of Premature-miR-181b by the Translin/Trax RNase Increases Vascular Smooth Muscle Cell Stiffness

Abstract

[Figure: see text].

Keywords: aorta; cardiovascular diseases; microRNA degradation; risk factors; vascular smooth muscle; vasopressin.

Figure 1.. AVP triggers degradation of pre-miR-181b in VSMCs and increases their stiffness.

(A) Serum AVP levels are elevated in mice maintained on HSW, rather than normal water (NW) for three weeks. (*; p<0.05) (B – C) Treatment of VSMCs with AVP (100 nM) for 24 hours reduces levels of both pre-miR-181b and miR-181b. (****; p<0.0001) (D) This AVP treatment also increases individual cell stiffness as measured by OMTC. (***; p<0.001, n>75 cells) (E) This AVP treatment does not alter levels of two control miRNAs, let-7a and miR-126-3p. (F) Angiotensin II (Ang-II) does not mimic the effect of AVP on levels of pre-miR-181b and miR-181b.

Figure 2.. Deletion or inactivation of the TN/TX complex abolishes effects of AVP on pre-miR-181b, miR-181b levels, and VSMC stiffness.

AVP treatment does not reduce levels of pre-miR-181b (A) or miR-181b (B) in VSMCs harvested from either Tsn^−/− mice or mice that are homozygous for the TX(E126A) point mutation that inactivates TN/TX RNase activity. (C) AVP’s ability to increase cell stiffness in VSMCs harvested from WT mice is also absent in VSMCs isolated from Tsn^−/− or TX(E126A) mice. In panel A, n=6 per group, WT CON vs WT AVP: ****, p <0.0001; WT CON vs TN KO CON: ****, p<0.0001; WT CON vs TXE126A CON: ***, p<0.001. Data analyzed by 2 way ANOVA followed by Tukey’s post-hoc analysis. In panel B, n=6 per group, WT CON vs WT AVP: ****, p <0.0001; WT CON vs TN KO CON: ****, p<0.0001; WT CON vs TXE126A CON: **, p<0.01. In panel C, n>90 cells per group, *, p<0.05 by 2 way ANOVA. followed by Tukey’s post-hoc analysis.

Figure 3.. Transfection of VSMCs with miR-181b blocks ability of AVP to reduce miR-181b levels and increase VSMC cell stiffness.

(A) VSMCs were transfected with either a miR-181b mimic (miR-181b) or scrambled (Scr) control and then treated with AVP or control media. Under these conditions, AVP reduces levels of pre-miR-181b, consistent with the ability of TN/TX to cleave a subset of pre-miRNAs in vitro (B) As pre-miR-181b is the precursor of mature miR-181b, its degradation by TN/TX in response to AVP stimulation in cells transfected with the scrambled (Scr) oligomer leads to decreased levels of endogenous mature miR-181b. However, transfection with a synthetic miR-181b mimic bypasses the TN/TX degradation pathway. Thus, transfection with the miR-181b mimic, but not the scrambled oligomer (Scr), elevates miR-181b levels and blocks the ability of AVP to decrease them. (C) Transfection with the miR-181b mimic oligo, but not the scarmbled control (Scr), also blocks the ability of AVP to increase cell stiffness. In panels (A) and (B), pre-miR-181b and miR-181b levels are normalized to the average value measured in VSMC transfected with miR-181b and treated with control media to emphasize the effect of miR-181b transfection. In panel A, n=5, Scr Control vs Scr AVP: ***, p <0.001; miR-181b Control vs miR-181b AVP, *, p<0.05. In panel B, n=5, Scr Control vs Scr AVP: ****, p <0.0001; Scr control vs miR-181b control: ****, p<0.0001. In panel C, n>75 cells per group, ****, p<0.0001.

Figure 4.. TGF-β mediates increased VSMC stiffness elicited by AVP.

(A) AVP treatment increases levels of TGF-beta in the culture media in VSMCs isolated from WT mice. This effect is absent in VSMCs harvested from TN KO or TX(E126A) mice. WT CON vs WT AVP: ****, p<0.0001; WT AVP vs TN KO AVP: ****, p<0.0001; WT AVP vs TXE126A AVP, ***, p<0.001. (B) Addition of neutralizing antibodies to TGF-β to VSMC culture media blocks the ability of AVP to increase cell stiffness. Vehicle control vs vehicle AVP: **, p<0.01, n>55 cells per group.

Figure 5.. Inactivation of the TN/TX complex in vivo blocks arterial stiffening in HSW-treated mice.

(A) At weeks 2 and 3 following onset of HSW ingestion, PWV is higher in WT than TX(E126A) mice. (n=4 mice per group; **, p<0.01; *, p<0.05. (B) Pulse pressure measured in the same cohort of mice is reduced in TX(E126A) mice at week 3 following onset of HSW ingestion. (**, p<0.01) (C) Aortic stiffness assayed by tensile testing of aortic rings harvested following HSW treatment is reduced in TX(E126A) mice, ***, p<0.001. Dotted lines represent +/− SEM.

Figure 6.. Inactivation of the TN/TX complex in vivo blocks age-associated aortic stiffening.

(A) To monitor development of aortic stiffness, PWV was measured in a cross-sectional study of young (4-6 months) and old (15-18 months) WT and TXE126A mice. WT Young vs WT Old: ****, p<0.0001; WT Old vs TXE126A Old: ****, p<0.0001, n=6 per group. (B-E) A longitudinal cohort study of WT and TXE126A mice shows divergence of aortic stiffness at 13 months based on (B) in vivo (PWV) (13 month WT vs. 13 month TXE126A: ****, p<0.0001; 13 month WT vs 5 month WT, ****, p<0.0001) and (C) in vitro (aortic ring stress-strain measures) assays. WT vs TX(E126A): ****, p<0.0001; Dotted lines represent +/− SEM. Pre-miR-181b (D) and miR-181b (E) levels are significantly elevated in TXE126A mice compared to WT at 13 months of age. *, p<0.05; **, p<0.01.