Deletion of the microRNA-degrading nuclease, translin/trax, prevents pathogenic vascular stiffness

Abstract

Vascular stiffness plays a key role in the pathogenesis of hypertension. Recent studies indicate that the age-associated reduction in miR-181b levels in vascular smooth muscle cells (VSMCs) contributes to increased vascular stiffness. As these findings suggest that inhibiting degradation of miR-181b might prevent vascular stiffening, we have assessed whether the microRNA-degrading translin/trax (TN/TX) complex mediates degradation of miR-181b in the aorta.We found that TN^-/- mice display elevated levels of miR-181b expression in the aorta. Therefore, we tested whether TN deletion prevents vascular stiffening in a mouse model of hypertension, induced by chronic high-salt intake (4%NaCl in drinking water for 3 wk; HSW). TN^-/- mice subjected to HSW stress do not show increased vascular stiffness, as monitored by pulse wave velocity and tensile testing. The protective effect of TN deletion in the HSW paradigm appears to be mediated by its ability to increase miR-181b in the aorta since HSW decreases levels of miR-181b in WT mice, but not in TN KO mice. We demonstrate for the first time that interfering with microRNA degradation can have a beneficial impact on the vascular system and identify the microRNA-degrading TN/TX RNase complex as a potential therapeutic target in combatting vascular stiffness.NEW & NOTEWORTHY While the biogenesis and mechanism of action of mature microRNA are well understood, much less is known about the regulation of microRNA via degradation. Recent studies have identified the protein complex, translin(TN)/trax(TX), as a microRNA-degrading enzyme. Here, we demonstrate that TN/TX is expressed in vascular smooth muscle cells. Additionally, deletion of the TN/TX complex selectively increases aortic miR-181b and prevents increased vascular stiffness caused by ingestion of high-salt water. To our knowledge, this is first report describing the role of a microRNA RNAse in cardiovascular biology or pathobiology.

Keywords: hypertension; miR-181b; miRNA-degradation, translin/trax complex, vascular stiffness.

Fig. 1.

Translin/trax (TN/TX) complex downregulates miR-181b in the aortic vascular smooth muscle cells (VSMCs). A: quantitative PCR (SYBR) analysis of TN and TX mRNAs in total RNA isolated from intact and denuded aortas. TN and TX expression was normalized to GAPDH. Vascular endothelial (VE)-cadherin expression was measured as a positive control of the denuded aorta. The means of the values obtained from intact tissue were set to 1.0; mean values obtained from denuded tissue are presented as the fold change relative to intact tissue. B: Scatterplot was generated by comparing TN knockout (TN^−/−) aorta miRNA-sequencing data with wild-type (WT) littermate aorta data. C: reads per million (RPM) values were calculated from the miRNA-sequencing data from aorta samples from WT and TN^−/− mice. The graph presents RPM valuses for the 15 most abundant miRNAs in aorta. D: RPM values were calculated from the RNA-sequencing data from aorta samples from WT and TN^−/− mice (n = 3). qPCR analysis of pre-miR-181b (E) and miR-181b (F) expression in total RNA from the aortas of WT and TN^−/− mice (n = 4 or 5). miRNA expression was normalized to SNORD61 and then normalized to WT expression. Values shown in all panels are means ± SE; *P < 0.05 and ***P < 0.001.

Fig. 2.

High-salt water (HSW) stress increases vascular stiffness. A: pulse wave velocity (PWV) was measured weekly in wild-type (WT) mice given normal water (NW) or HSW (n = 6). Data presented in A were analyzed by 2-way ANOVA with repeated measures, which showed significant effects of treatment (P < 0.0001) and time (P < 0.01), as well as a significant interaction between these variables (P < 0.01). Post hoc Bonferroni’s test showed significant differences at time points denoted by ****P < 0.0001 B: tail-cuff based measurement of systolic blood pressure after 3 wk of HSW (n = 13) and NW (n = 8). Tensile testing was performed on thoracic aortas that were either intact (C) or decellularized (D) from WT animals given NW or HSW (n = 5–6 mice with 3 to 4 aortic rings per mouse). The curved line plots the mean values at each value of strain. SE is presented as dots located above and below the curve. Data were analyzed by 2-way ANOVA with repeated measures. Analysis of data presented in C showed significant effects of treatment (P < 0.02) and strain (P < 0.0001), as well as a significant interaction between these variables (P < 0.0001). Similarly, analysis of data presented in D showed significant effects of treatment (P < 0.05) 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 < 0.01 for C and P < 0.05 for D as shown in bar graph inserts. Values shown are means ± SE; *P < 0.05, **P < 0.01 and ****P < 0.0001.

Fig. 3.

Translin (TN) deletion blocks increase in vascular stiffness and decrease in miR-181b expression produced by high-salt water (HSW) stress. A: weekly pulse wave velocity (PWV) measurements comparing responses of TN knockout (TN^−/−) and control [wild-type (WT)] (n = 8) mice treated with HSW. Analysis of data presented in A showed significant effects of genotype (P < 0.05) and time (P < 0.01), as well as a significant interaction between these variables (P < 0.05). Post hoc Bonferroni’s test showed significant differences at time points denoted by *P < 0.05. B: tailoremetric measurement of systolic blood pressure after 3 wk of normal water (NW) and HSW in TN^−/− mice C: tensile testing was performed on the thoracic aorta from WT and TN^−/− mice treated with HSW (n = 6 animals with 3 to 4 aortic rings per animal). Data were analyzed by 2-way ANOVA with repeated measures, which showed significant effects of genotype (P < 0.02) 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 < 0.02. Quantitative PCR data examining pre-miR-181b (D) and miR-181b (E) expression in total RNA from aortas of WT or TN^−/− mice treated with NW or HSW (n = 3). Pre- or mature miR-181b expression was normalized to SNORD61 and then to the WT NW group. F: Let-7a and miR-126-3p in total RNA normalized to SNORD61 from aortas of WT mice treated with NW or HSW (n = 3). Values are means ± SE. *P < 0.05, ***P < .001, #P < 0.01 WT + HSW vs. TN^−/− + HSW.

Fig. 4.

High-salt water (HSW) effects on serum TGF-β1 and aortic wall collagen content and media area. A: serum TGF-β1 levels in wild-type (WT) and translin knockout (TN^−/−) mice when exposed to normal water (NW) and HSW (n = 6–8). Because of unequal variance across these groups, the data were rank transformed and then analyzed by 2-way ANOVA using the general linear model. Post hoc testing using the Holm-Sidak method showed a significant difference between the WT + HSW and TN^−/− + HSW groups (P < 0.001). B and C: Masson trichrome staining. Collagen content of aortic rings expressed as a percentage of total ring area in WT and TN^−/− exposed to NW and HSW. D: aortic media area in WT and TN^−/− mice exposed to NW or HSW. Values are means ± SE. *P < 0.05, ***P < .001.

Fig. 5.

High-salt water (HSW) upregulates trax (TX) expression. Quantitative PCR data examining TN (A) and TX mRNA (B) levels in wild-type (WT) mouse aorta with normal water (NW) or HSW (n = 3). The expression data were normalized to β-actin. C: Western blot analysis of TN and TX expression in aortic tissue from WT mice given access to NW or HSW (n = 3). TN and TX expression was normalized to α-tubulin. C, right: quantification of band intensities. Values are means ± SE. *P < 0.05.

Fig. 6.

Working model of vascular stiffness regulation by miR-181b and translin/trax (TN/TX) complex. Left: chronic exposure of wild-type mice to high-salt water (HSW) leads to increased vascular stiffness. This treatment elicits decreased levels of pre-miR-181b and mature miR-181b, suggesting that increased activity of TN/TX RNase mediates degradation of pre-miR-181b and/or mature miR-181b. Consistent with this model, chronic HSW treatment does not elevate aortic stiffness in TN knockout (TN KO) mice, which display elevated levels of pre-miR-181b and miR-181b (right). Absence of TN/TX in TN KO mice is indicated by an oval containing gray diagonal stripes.