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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Hypertension. 2021 Jul 26;78(3):831–839. doi: 10.1161/HYPERTENSIONAHA.120.16690

Degradation of premature-miR-181b by the translin/trax RNase increases vascular smooth muscle cell stiffness

Eric Tuday 1,2, Mitsunori Nakano 3, Kei Akiyoshi 3, Xiuping Fu 4, Aparna P Shah 4, Atsushi Yamaguchi 5, Charles Steenbergen 6, Lakshmi Santhanam 3, Steven S An 7,8, Dan Berkowitz 9, Jay M Baraban 4,10,*, Samarjit Das 3,6,*
PMCID: PMC8363557  NIHMSID: NIHMS1719566  PMID: 34304585

Abstract

Large artery stiffness (LAS) is a major risk factor underlying cardiovascular disease. However, the molecular mechanisms driving this pathological process are poorly understood. Previous studies indicate that the age-associated decline of miR-181b levels can accelerate aortic stiffening by activating TGF-β signaling. Here, we studied the physiological role of miR-181b in mediating arginine vasopressin (AVP)-induced stiffening of vascular smooth muscle cells (VSMCs) isolated from aorta. We found that AVP treatment increases VSMC stiffness and causes marked reductions in both pre-miR-181b and miR-181b expression. Transfecting VSMCs with a miR-181b mimic abolishes AVP-induced stiffening, indicating that this stiffening response is dependent on AVP’s ability to reduce miR-181b levels. In addition, deletion of translin or inactivation of the translin/trax (TN/TX) RNase prevents the AVP-induced decrease in pre-miR-181b/miR-181b levels and VSMC stiffening, indicating that these effects are mediated by this microRNA-degrading enzyme. Interestingly, AVP exposure increases extracellular TGF-β levels in a TN/TX-dependent manner, and pre-treatment of VSMCs with TGF-β neutralizing antibodies inhibits AVP-induced stiffness. Lastly, we have ascertained that age-associated aortic stiffening in vivo is prevented in mice homozygous for the TX(E126A) point mutation, which abolishes TN/TX RNase activity. Taken together, these findings provide compelling evidence that TN/TX RNase activity plays a critical role in regulating VSMC stiffness via degradation of pre-miR-181b and/or TGF-β pathway activation. Our findings also indicate that therapeutic strategies capable of blocking TN/TX-mediated reductions in miR-181b levels may confer protection against LAS and associated cardiovascular diseases.

Keywords: microRNA, miR-181b, Translin/Trax, Vascular stiffness, TGF-β, miRNA degradation

Summary

In this study, we have used vascular smooth muscle cell cultures to demonstrate that TN/TX activation elicits cell stiffening by decreasing miR-181b levels and promoting TGF-β signaling. Furthermore, genetic inactivation of the TN/TX RNase in vivo blocks HSW-induced or age-associated aortic stiffening.

Introduction

Identification of large artery stiffness (LAS) as an independent risk factor for cardiovascular disease-associated morbidity and mortality has heightened interest in understanding the molecular mechanisms that drive this process 1–4 LAS has also been implicated in the development of many age-associated co-morbidities such as coronary artery disease, congestive heart failure, chronic kidney disease, and dementia 2, 3, 5–12 This occurs, in part, as the result of altered hemodynamics causing damaging pressure gradients in the microvascular beds 1, 5, 13. Interventions to date have had limited success in either treating or preventing age-associated increases in LAS, however, emphasizing the need for new strategies to address this endemic risk factor 9, 14–17.

MicroRNAs (miRs) have emerged as ubiquitous regulators of protein translation. These small, ~20-22 nucleotide RNA fragments inhibit translation of their target mRNAs by binding to complementary sequences in their 3’-UTR 18, 19. The canonical pathway for biogenesis of miRs starts with transcription from portions of the genome that were considered “junk” DNA. This primary transcript is cleaved by Drosha into premature miRs (pre-miRs) that are then processed further by Dicer to generate mature miRs 18, 19. It is now clear that the miR signaling network plays a key role in fine tuning spatial and temporal control of translation in response to developmental and environmental signals.

As dysregulation of miR signaling contributes to a wide range of pathologic processes, recent studies have focused attention on the role of miRs in regulating vascular smooth muscle cell (VSMC) function and the pathophysiology of hypertension 20–24. Based on this research, the following miRs have been implicated in arterial stiffening: miR-76525, miR-118526, miR-21 27, and miR-181b. Among these, miR-181b has emerged as an attractive candidate for mediating age-related changes in vascular stiffness 18. The observation that aortic expression of miR-181b decreases with age prompted an examination of the impact of deleting the miR-181a/b-1 locus, a manipulation that causes near complete loss of miR-181b expression in mouse aorta 18. Deletion of this locus accelerates the development of aortic stiffness, which is accompanied by heightened activation of the TGF-β pathway, a predicted target of miR-181b 18. In addition, development of aortic stiffness in these mice is accompanied by increased deposition of collagen in the extracellular matrix (ECM) 18. Taken together, these findings indicate that: 1) age-dependent reduction in miR-181b levels in the aorta plays a pivotal role in age-associated increases in LAS, and 2) interventions capable of preserving miR-181b levels might combat stiffening.

Recent studies have identified the translin/trax (TN/TX) RNase, as a microRNA-degrading enzyme that targets a small subpopulation of pre-miRs 28. Degradation of these pre-miRNAs by TN/TX prevents them from being processed to mature miRs by Dicer, thereby decreasing levels of the corresponding mature miRs. Conversely, deletion of translin (Tsn), which abolishes TN/TX RNase activity, leads to elevation of pre-miRs targeted by this complex, as well as their mature forms. Previously, we have ascertained that Tsn −/− mice display selective elevation of both pre-miR-181b and mature miR-181b levels in the aorta. Furthermore, we have found that, in contrast to WT mice, these mice do not develop aortic stiffening when placed on a high salt-water (HSW) regimen 19. Thus, these studies suggest that the protective effect conferred by Tsn deletion is mediated by elevated miR-181b levels. To test this hypothesis, we investigated the role of TN/TX and miR-181b in regulating stiffness of VSMCs isolated from aorta.

Materials and methods

Data created for the study are available in a persistent repository.

Animals.

We used male wild-type (WT), Tsn−/−, and TX(E126A) mice to conduct the experiments in this study. Information about the origin of these transgenic lines are provided in the Supplemental Information section. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Johns Hopkins University.

Individual cell stiffness measurements.

Optical magnetic twisting cytometry (OMTC) was utilized to measure individual cell stiffness 29–31 as described in the supplemental information section.

Non-Invasive Pulse Wave Velocity (PWV) Measurements.

A high-frequency, high-resolution Doppler spectrum analyzer (DSPW, Indus Instruments, TX) was used under isoflurane anesthesia 18, 19.

RNA Isolation and qPCR.

Total RNA and miRNA-enriched fractions were isolated from aortic tissue using the RNeasy and miRNeasy kits, respectively (Qiagen, Valencia, CA, USA). The detailed protocol is provided in the Supplemental Information section.

Statistical analysis.

The results are presented as means +/− SE. For multiple comparisons, one-way or two-way analysis of variance (ANOVA) and the Bonferroni or Holm-Sidak post hoc tests were used. P < 0.05 was considered statistically significant. All analyses were performed using Prism 8 (GraphPad Software) or SigmaStat (San Jose, CA).

Results

Arginine vasopressin (AVP) triggers degradation of pre-miR-181b and increases VSMC stiffness

To study the hypothesized link between TN/TX, miR-181b and VSMC stiffness, we explored the possibility that AVP might trigger pre-miR-181b degradation by activating TN/TX. We focused on AVP as a leading candidate because mice subjected to 3 weeks of HSW, a paradigm that increases aortic stiffness, display increased serum AVP levels and lower levels of miR-181b in aorta (Fig. 1A). We examined the impact of AVP on miR-181b levels and cell stiffness using the A7r5 rat vascular smooth muscle cell line. We found that addition of AVP (100 nM) to the culture media for 24 hrs lowers levels of both pre-miR-181b (Fig. 1B) and mature miR-181b (Fig. 1C). In addition, AVP treatment increases VSMC stiffness compared to the vehicle treated group (Fig. 1D).

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

Figure 1.

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(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.

To assess whether AVP treatment affects miR-181b selectively, we also checked its effect on levels of let-7a and miR-126-3p, two miRNAs that are abundant in the aorta. We found that AVP treatment does not affect let-7a or miR-126-3p levels (Fig. 1E). To check the specificity of AVP’s ability to decrease levels of pre-miR-181b and miR-181b, we also examined whether Ang II, which also promotes smooth muscle contraction, mimics AVP’s effects in these cells. For these studies, we incubated A7r5 cells with Ang II (10nM through 1 μM) for 24 hr. However, unlike AVP, Ang II treatment does not alter pre-miR-181b and miR-181b expression in these VSMCs (Fig. 1F). Of note, normal human plasma contains 5.91 + 0.83 pM AVP 32 and mean plasma angiotensin II level is 18.4 ± 3.3 pM 33.

VSMC responses to AVP are mediated by the TN/TX RNase

To check if the TN/TX microRNA-degrading enzyme mediates AVP-induced degradation of pre-miR-181b, we tested the effect of AVP on VSMCs isolated from the aorta of Tsn−/− mice, which lack the TN/TX complex. Furthermore, since it is conceivable that deletion of Tsn may also have other biological effects besides eliminating the TN/TX complex, we also tested the impact of AVP on aortic VSMCs isolated from mice that are homozygous for a point mutation in TX, E126A, that inactivates TN/TX RNase activity without altering TN or TX protein levels 34. In VSMCs isolated from the aorta of either the Tsn−/− or TX(E126A) mice, AVP treatment does not trigger reductions in levels of pre-miR-181b (Fig. 2A) or miR-181b (Fig. 2B). In contrast, AVP decreases levels of both pre-miR-181b (Fig. 2A) and miR-181b (Fig. 2B) expression in VSMCs isolated from WT aortas. In addition, AVP treatment does not increase cellular stiffness of VSMCs isolated from Tsn−/− or TX(E126A) aorta (Fig. 2C).

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

Figure 2.

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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.

As Tsn deletion and the TX(E126A) point mutation abolish TN/TX RNase activity throughout development, it is conceivable that the blockade of AVP’s effects observed in VSMCs harvested from mature mice might be due to indirect, compensatory effects elicited by these constitutive genetic alterations. Accordingly, we confirmed that acute knockdown of TX in A7r5 cells also blocks both these effects of AVP (Supplemental Fig. S1).

AVP-induced reduction in miR-181b levels mediates cellular stiffening

As AVP reduces miR-181b levels and increases cell stiffness, we wanted to check if these effects are causally linked. To this end, we monitored AVP’s effect on stiffness of A7r5 cells that had been transfected with an LNA oligo that mimics mature miR-181b, thereby bypassing AVP’s ability to reduce endogenous miR-181b levels. As shown in Fig. 3A, AVP treatment downregulates pre-miR-181b levels in both the scrambled- and miR-181b-transfected groups, as expected. However, miR-181b levels remain elevated in cells transfected with the miR-181b mimic. Of note, this treatment blocks the ability of AVP to induce cell stiffening (Fig. 3C) demonstrating that this response is critically dependent on AVP’s ability to reduce miR-181b levels.

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

Figure 3.

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(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 28 (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.

Activation of the TGF-β pathway by AVP mediates increased stiffness

In previous studies 18, we found that deletion of miR-181b leads to increased aortic stiffness that is accompanied by augmented TGF-β signaling and increased deposition of extracellular collagen. Therefore, we tested whether AVP-induced increases in cell stiffness might also be dependent on increased TGF-β signaling. To examine this possibility, we measured extracellular TGF-β abundance in the culture media from primary VSMCs isolated from WT, Tsn−/− and TX(E126) mouse aortas following AVP treatment. Consistent with our previous finding 18, AVP treatment significantly increases TGF-β levels in the media of VSMCs isolated from WT aortas, but not from those isolated from aortas of either Tsn−/− or TX(E126) mice (Fig. 4A). Furthermore, to test whether AVP-induced stiffening is mediated by TGF-β, we checked the effect of adding TGF-β neutralizing antibodies to the culture media on VSMC stiffness. Primary VSMCs were treated with AVP in the presence of TGF-β antibodies or control rabbit serum. AVP increases VSMC stiffness in the presence of control rabbit serum (Fig. 4B). However, TGF-β neutralizing antibody abolishes the ability of AVP to increase VSMC stiffness (Fig. 4B).

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

Figure 4.

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(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.

Inactivation of TN/TX RNase activity protects from aortic stiffening in vivo

Our previous studies indicate that the age-associated decline of miR-181b expression in VSMCs of the aorta promotes its stiffening 18. Furthermore, we also found that Tsn−/− mice do not develop increased aortic stiffness when subjected to the HSW paradigm 19. Therefore, we checked whether mice homozygous for the TX(E126A) mutation, which abolishes TN/TX RNase activity, are protected from aortic stiffening elicited by HSW treatment or associated with aging.

In the first approach, pulse wave velocity (PWV) in the descending aorta was measured under baseline conditions and then weekly after switching the mice to HSW (Fig 5A). To check whether switching the mice to HSW might have non-specific adverse effects, we confirmed that this paradigm does not affect the body weight of either WT or TX (E126A) mice (Supplemental Fig. S2). While WT mice display increased PWV at 2 and 3 weeks, as expected, TX(E126A) mice do not (Fig. 5A). Monitoring of pulse pressure at weekly intervals during the HSW paradigm revealed that WT mice develop increased pulse pressure at week 3, while TX(E126A) mice do not (Figure 5B). Furthermore, consistent with the PWV values, tensile testing of aortic rings isolated from these mice confirmed that TX(E126A) mice have lower aortic stiffness than WT mice following exposure to HSW (Figure 5C).

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

Figure 5.

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(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.

To assess whether TX(E126A) mice are protected from developing increased aortic stiffness with aging as found in WT mice, we compared PWV in mutant and WT littermates at 4-6 months and at 17-19 months (Figure 6A). The younger WT and TX(E126A) mice did not differ in PWV. However, the PWV of the older WT mice was higher than that of the older TX(E126A) mice.

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

Figure 6.

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(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.

Based on the cross-sectional observations reported in Figure 6A, we sought to determine if the protective effect displayed by TX(E126A) mice is also apparent earlier than 17-19 months. Recent studies have reported that mice lacking one allele of the lysyl oxidase-like 2 (Loxl2) gene show decreased PWV compared to WT mice at 12-15 months of age 35. Accordingly, we compared the PWV of WT and TX(E126A) mice at 13 months. WT mice display increased PWV compared to their baseline values taken at 5 months of age or to 13 month old TX(E126A) mice (Figure 6B). Tensile testing of rings harvested from the descending aorta of these mice at 13 months of age showed a leftward shift in the stress-strain relationship of the WT mice compared to TX(E126A) mice, consistent with the PWV measurements (Figure 6C). A clear prediction of our working model is that protection from aortic stiffening exhibited by TX(E126A) mice is due to elevated levels of miR-181b. Accordingly, we confirmed that levels of both pre-miR-181b and mature miR-181b are elevated in the aorta of TX(E126A) mice compared to WT mice at 13 months of age (Figure 6D and E). Lastly, since it is known that aged WT mice display increased aortic diameter and wall thickness36, we examined whether the TX(E126A) mutation affects aortic diameter and wall thickness in aged mice. We found that our cohort of WT aged mice fall on the spectrum of previously reported age-related changes in aortic diameter and wall thickness36. However, the aged TX(E126A) mice have reduced overall aortic diameter and wall thickness compared to WT mice (Supplemental Table 1).

Discussion

LAS is a major, independent risk factor for a wide range of cardiovascular diseases including isolated systolic hypertension, “an endemic condition responsible for a large proportion of the global burden of cardiovascular morbidity and mortality” 1. As there is a marked increase in LAS with aging, this risk factor presents a major public health challenge in this expanding population. The pathophysiological mechanisms underlying isolated systolic hypertension are distinct from those causing essential hypertension. Nevertheless, both forms of hypertension are currently treated with anti-hypertensive medications designed for essential hypertension, even though their efficacy in treating isolated systolic hypertension is limited and exposes these patients to potentially serious complications resulting from orthostatic hypotension. The current study provides compelling evidence that inhibitors of the TN/TX microRNA-degrading enzyme represent a novel therapeutic approach for combatting LAS by elevating levels of miR-181b.

In previous in vivo studies, we found that Tsn−/− mice: 1) are protected from developing aortic stiffening induced by chronic treatment with HSW, and 2) do not display reduced miR-181b levels following the HSW regimen 19. Furthermore, we found that mice lacking the miR-181a1/b1 locus, which is the major source of miR-181b in aorta, display accelerated development of aortic stiffening that is associated with augmented TGF-β signaling 18. Integrating these results suggested that: 1) the protection conferred by Tsn deletion is mediated by blocking the decline in miR-181b levels induced by HSW, and 2) the increased aortic stiffness induced by reduced miR-181b levels is mediated by elevated TGF-β signaling. Accordingly, the goal of this study was to test key aspects of this working model. To this end, we found that incubation of VSMCs with AVP triggers degradation of miR-181b and increases individual cell stiffness. Then, we used this in vitro model to confirm the causal links between TN/TX, miR-181b, TGF-β signaling and cell stiffness posited by our working model (Supplemental Fig. S4).

Since it is unclear whether AVP-induced VSMC stiffness represents a bona fide model of aortic stiffness induced by HSW or associated with aging in vivo, we have confirmed the prediction that inactivation of TN/TX confers protection from aortic stiffness in vivo in both these paradigms. As Asada et al.28 have demonstrated that the TN/TX RNase is “druggable”, these findings suggest that selective inhibitors of TN/TX RNase activity may have therapeutic potential in preventing, arresting or even reversing, the progression of LAS. It is important to point out that, similar to Tsn−/− mice, mice that are homozygous for the TX(E126A) mutation display robust adiposity, in the context of normal body weight. However, recent studies demonstrated that global deletion of Tsn in adulthood does not elicit abnormal adiposity 34. Thus, treatment with an inhibitor of TN/TX in adulthood would not be expected to increase adiposity.

Our finding that AVP reduces pre-miR-181b and miR-181b levels in a TN/TX-dependent fashion is consistent with prior studies suggesting that Gq, which mediates downstream effects of AVP 37, plays a key role in regulating TN/TX RNase activity 37 Those studies suggest that PLCβ inhibits TN/TX activity by binding to TX. In this scenario, activation of Gq triggers translocation of PLCβ from the TN/TX complex to Gq, which relieves its inhibition of TN/TX. Accordingly, it will be interesting in future studies to test this model of TN/TX activation in VSMCs. Furthermore, elucidating the signaling pathway linking AVP stimulation to TN/TX activation may shed light on the unexpected observation that Ang II, which shares some of the signaling properties of AVP 38, 39, does not mimic AVP’s ability to activate TN/TX.

Our current study fits well with our previous in vivo studies, which indicated that reduced levels of miR-181b in aorta VSMCs elicit aortic stiffening via enhancing TGF-β signaling and its known downstream effects on vascular remodeling 18. Our supplementary data (Table 1, Fig. S3) support this inference by indicating that aged TX(E126A) mice display favorable vessel characteristics. In addition to morphological analysis demonstrating reduced aortic diameter and wall thickness, we also conducted pilot histologic analysis of aortic rings, which, although preliminary, suggest that aged TX(E126A) mice display reduced medial collagen content and preserved elastin structure compared to aged WT mice. However, it will be important to identify the molecular mechanisms that link miR-181b to TGF-β signaling and fully characterize the impact of the TX(E126A) mutation in aged mice. In general, miRs exert their cellular effects by suppressing translation of selected mRNAs by binding to their 3’UTR 40. In previous studies, we identified one candidate target mRNA, TGF-βi, a poorly characterized transcript that is induced in response to TGF-β stimulation 18. However, it is unclear if this target accounts for the increase in extracellular levels of TGF-β1 induced by AVP in vitro or by reduced miR-181b levels in vivo 18. Accordingly, our findings suggest that this in vitro model of VSMC stiffening may be useful for identifying transcripts that undergo increased translation in response to reduced miR-181b levels and determine if they mediate the AVP-induced increase in extracellular TGF-β abundance.

In conclusion, by investigating the signaling pathway that links AVP to increased VSMC stiffness in vitro, we have confirmed key aspects of our working model (Supplemental Fig. S4): activation of TN/TX triggers degradation of pre-miR-181b; the ensuing decline in miR-181b abundance elicits an increase in extracellular TGF-β levels which augments cell stiffening. Furthermore, we have validated the translational relevance of this in vitro model by confirming that inactivation of TN/TX in vivo prevents HSW-induced or age-associated elevation of aortic stiffness.

Supplementary Material

Online supplemental

Perspectives.

Although dysregulation of microRNAs has been implicated as a causal factor in a wide range of pathological conditions, it has been challenging to develop therapeutic strategies to remedy these abnormalities. This study focuses attention on the feasibility of a novel approach, i.e. inhibition of microRNA-degrading enzymes. As reduced levels of a specific microRNA, miR-181b, has been implicated in the pathophysiology of large artery stiffness, a major cause of cardiovascular morbidity, we reasoned that inhibition of the translin/trax microRNA-degrading enzyme, which targets the precursor form of this microRNA, might be effective in combatting this condition. By using an in vitro model of vascular smooth muscle stiffness, we demonstrate that arginine vasopressin increases the stiffness of individual vascular smooth muscle cells by decreasing levels of miR-181b, an effect that is mediated by the translin/trax microRNA-degrading enzyme. Furthermore, we confirm that this microRNA-degrading enzyme plays a critical role in the development of age-associated vascular stiffening, in vivo. Thus, taken together, these studies provide compelling evidence that inhibition of a microRNA-degrading enzyme, the translin/trax RNase, provides a feasible strategy to reverse pathological changes linked to microRNA dysregulation. Accordingly, these findings provide a firm basis for future studies evaluating the utility of this novel approach in treating large artery stiffness. Furthermore, these findings suggest that this approach, inhibition of microRNA-degradation, may be extended to treating other conditions linked to microRNA dysregulation.

Novelty and Significance.

What is new?

Deletion of the translin/trax (TN/TX) microRNA-degrading enzyme that degrades pre-miR-181b, protects against aortic stiffening induced by chronic treatment with high salt water (HSW).

What is relevant?

Novel therapeutic approaches are needed to combat development of large artery stiffness (LAS), an endemic, age-associated condition that is responsible for a large proportion of cardiovascular morbidity and mortality globally. Recent studies implicate the age-dependent decline of miR-181b levels in aorta in mediating LAS by enhancing TGF-β signaling.

Acknowledgments and Sources of Funding

This work was supported by grants from the MSCRF, Mscrfd-4313, U54AG062333 and U18TR003780 (S.D.), NIH P01 HL114471 and R01 DK035385 (S.S.A), Johns Hopkins Synergy Award (J.M.B), Magic That Matters (E.T., J.M.B., and S.D.), NIH T32, T32HL007227 (E.T.), and Stimulating and Advancing ACCM Research (S.D.). We thank Arie Horowitz for commenting on this manuscript.

Abbreviation

AVP

Arginine Vasopressin

miRNA

microRNA

Pre-miR

Premature miRNA

PWV

Pulse Wave Velocity

TGF-β

Transforming Growth Factor-beta

TN/TX

Translin/Trax

VSMC

Vascular smooth muscle cell

OMTC

Optical magnetic twisting cytometry

References

  • 1.Chirinos JA, Segers P, Hughes T, Townsend R. Large-artery stiffness in health and disease: Jacc state-of-the-art review. J Am Coll Cardiol. 2019;74:1237–1263 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Mitchell GF, Hwang SJ, Vasan RS, Larson MG, Pencina MJ, Hamburg NM, Vita JA, Levy D, Benjamin EJ. Arterial stiffness and cardiovascular events: The framingham heart study. Circulation. 2010;121:505–511 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.O’Rourke M Arterial stiffness, systolic blood pressure, and logical treatment of arterial hypertension. Hypertension. 1990;15:339–347 [DOI] [PubMed] [Google Scholar]
  • 4.O’Rourke MF, Nichols WW. Aortic diameter, aortic stiffness, and wave reflection increase with age and isolated systolic hypertension. Hypertension. 2005;45:652–658 [DOI] [PubMed] [Google Scholar]
  • 5.Briet M, Boutouyrie P, Laurent S, London GM. Arterial stiffness and pulse pressure in ckd and esrd. Kidney Int. 2012;82:388–400 [DOI] [PubMed] [Google Scholar]
  • 6.Group SMIftSR, Williamson JD, Pajewski NM, Auchus AP, Bryan RN, Chelune G, Cheung AK, Cleveland ML, Coker LH, Crowe MG, Cushman WC, Cutler JA, Davatzikos C, Desiderio L, Erus G, Fine LJ, Gaussoin SA, Harris D, Hsieh MK, Johnson KC, Kimmel PL, Tamura MK, Launer LJ, Lerner AJ, Lewis CE, Martindale-Adams J, Moy CS, Nasrallah IM, Nichols LO, Oparil S, Ogrocki PK, Rahman M, Rapp SR, Reboussin DM, Rocco MV, Sachs BC, Sink KM, Still CH, Supiano MA, Snyder JK, Wadley VG, Walker J, Weiner DE, Whelton PK, Wilson VM, Woolard N, Wright JT Jr., Wright CB. Effect of intensive vs standard blood pressure control on probable dementia: A randomized clinical trial. JAMA. 2019;321:553–561 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Group SR, Wright JT Jr., Williamson JD, Whelton PK, Snyder JK, Sink KM, Rocco MV, Reboussin DM, Rahman M, Oparil S, Lewis CE, Kimmel PL, Johnson KC, Goff DC Jr., Fine LJ, Cutler JA, Cushman WC, Cheung AK, Ambrosius WT. A randomized trial of intensive versus standard blood-pressure control. N Engl J Med. 2015;373:2103–2116 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Okamoto M, Nakamura F, Musha T, Kobayashi Y. Association between novel arterial stiffness indices and risk factors of cardiovascular disease. BMC Cardiovasc Disord. 2016;16:211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Raij L, Gonzalez-Ochoa AM. Vascular compliance in blood pressure. Curr Opin Nephrol Hypertens. 2011;20:457–464 [DOI] [PubMed] [Google Scholar]
  • 10.Townsend RR, Anderson AH, Chirinos JA, Feldman HI, Grunwald JE, Nessel L, Roy J, Weir MR, Wright JT Jr., Bansal N, Hsu CY, Investigators CS. Association of pulse wave velocity with chronic kidney disease progression and mortality: Findings from the cric study (chronic renal insufficiency cohort). Hypertension. 2018;71:1101–1107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Vlachopoulos C, Aznaouridis K, Stefanadis C. Prediction of cardiovascular events and all-cause mortality with arterial stiffness: A systematic review and meta-analysis. J Am Coll Cardiol. 2010;55:1318–1327 [DOI] [PubMed] [Google Scholar]
  • 12.Weber T, Auer J, O’Rourke MF, Kvas E, Lassnig E, Berent R, Eber B. Arterial stiffness, wave reflections, and the risk of coronary artery disease. Circulation. 2004;109:184–189 [DOI] [PubMed] [Google Scholar]
  • 13.Kucharska-Newton AM, Stoner L, Meyer ML. Determinants of vascular age: An epidemiological perspective. Clin Chem. 2019;65:108–118 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Agabiti-Rosei E, Porteri E, Rizzoni D. Arterial stiffness, hypertension, and rational use of nebivolol. Vasc Health Risk Manag. 2009;5:353–360 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Boutouyrie P, Lacolley P, Briet M, Regnault V, Stanton A, Laurent S, Mahmud A. Pharmacological modulation of arterial stiffness. Drugs. 2011;71:1689–1701 [DOI] [PubMed] [Google Scholar]
  • 16.Schiffrin EL. Circulatory therapeutics: Use of antihypertensive agents and their effects on the vasculature. J Cell Mol Med. 2010;14:1018–1029 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Williams B Evaluating interventions to reduce central aortic pressure, arterial stiffness and morbidity--mortality. J Hypertens. 2012;30Suppl:S13–18 [DOI] [PubMed] [Google Scholar]
  • 18.Hori D, Dunkerly-Eyring B, Nomura Y, Biswas D, Steppan J, Henao-Mejia J, Adachi H, Santhanam L, Berkowitz DE, Steenbergen C, Flavell RA, Das S. Mir-181b regulates vascular stiffness age dependently in part by regulating tgf-beta signaling. PLoS One. 2017;12:e0174108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Tuday E, Nomura Y, Ruhela D, Nakano M, Fu X, Shah A, Roman B, Yamaguchi A, An SS, Steenbergen C, Baraban JM, Berkowitz DE, Das S. Deletion of the microrna-degrading nuclease, translin/trax, prevents pathogenic vascular stiffness. Am J Physiol Heart Circ Physiol. 2019;317:H1116–H1124 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Albinsson S, Suarez Y, Skoura A, Offermanns S, Miano JM, Sessa WC. Micrornas are necessary for vascular smooth muscle growth, differentiation, and function. Arterioscler Thromb Vasc Biol. 2010;30:1118–1126 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Turczynska KM, Sadegh MK, Hellstrand P, Sward K, Albinsson S. Micrornas are essential for stretch-induced vascular smooth muscle contractile differentiation via microrna (mir)-145-dependent expression of l-type calcium channels. The Journal of biological chemistry. 2012;287:19199–19206 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Bhattachariya A, Dahan D, Turczynska KM, Sward K, Hellstrand P, Albinsson S. Expression of micrornas is essential for arterial myogenic tone and pressure-induced activation of the pi3-kinase/akt pathway. Cardiovascular research. 2014; 101:288–296 [DOI] [PubMed] [Google Scholar]
  • 23.Kang H, Hata A. Microrna regulation of smooth muscle gene expression and phenotype. Current opinion in hematology. 2012;19:224–231 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Carr G, Barrese V, Stott JB, Povstyan OV, Jepps TA, Figueiredo HB, Zheng D, Jamshidi Y, Greenwood IA. Microrna-153 targeting of kcnq4 contributes to vascular dysfunction in hypertension. Cardiovascular research. 2016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Liao YC, Wang YS, Hsi E, Chang MH, You YZ, Juo SH. Microrna-765 influences arterial stiffness through modulating apelin expression. Mol Cell Endocrinol. 2015;411:11–19 [DOI] [PubMed] [Google Scholar]
  • 26.Deng H, Song Z, Xu H, Deng X, Zhang Q, Chen H, Wang Y, Qin Y, Li Y. Microrna-1185 promotes arterial stiffness though modulating vcam-1 and e-selectin expression. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology. 2017;41:2183–2193 [DOI] [PubMed] [Google Scholar]
  • 27.Parthenakis F, Marketou M, Kontaraki J, Patrianakos A, Nakou H, Touloupaki M, Vernardos M, Kochiadakis G, Chlouverakis G, Vardas P. Low levels of microrna-21 are a marker of reduced arterial stiffness in well-controlled hypertension. Journal of clinical hypertension (Greenwich, Conn.). 2016 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Asada K, Canestrari E, Fu X, Li Z, Makowski E, Wu YC, Mito JK, Kirsch DG, Baraban J, Paroo Z. Rescuing dicer defects via inhibition of an anti-dicing nuclease. Cell Rep. 2014;9:1471–1481 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.An SS, Fabry B, Trepat X, Wang N, Fredberg JJ. Do biophysical properties of the airway smooth muscle in culture predict airway hyperresponsiveness? Am J Respir Cell Mol Biol. 2006;35:55–64 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.An SS, Kim J, Ahn K, Trepat X, Drake KJ, Kumar S, Ling G, Purington C, Rangasamy T, Kensler TW, Mitzner W, Fredberg JJ, Biswal S. Cell stiffness, contractile stress and the role of extracellular matrix. Biochem Biophys Res Commun. 2009;382:697–703 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhu W, Kim BC, Wang M, Huang J, Isak A, Bexiga NM, Monticone R, Ha T, Lakatta EG, An SS. Tgfbeta1 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness. Sci Rep. 2018;8:2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Pullan PT, Clappison BH, Johnston CI. Plasma vasopressin and human neurophysins in physiological and pathological states associated with changes in vasopressin secretion. J Clin Endocrinol Metab. 1979;49:580–587 [DOI] [PubMed] [Google Scholar]
  • 33.Schulz A, Jankowski J, Zidek W, Jankowski V. Absolute quantification of endogenous angiotensin ii levels in human plasma using esi-lc-ms/ms. Clin Proteomics. 2014;11:37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Fu X, Shah AP, Li Z, Li M, Tamashiro KL, Baraban JM. Genetic inactivation of the translin/trax microrna-degrading enzyme phenocopies the robust adiposity induced by translin (tsn) deletion. Mol Metab. 2020;40:101013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Steppan J, Wang H, Bergman Y, Rauer MJ, Tan S, Jandu S, Nandakumar K, Barreto-Ortiz S, Cole RN, Boronina TN, Zhu W, Halushka MK, An SS, Berkowitz DE, Santhanam L. Lysyl oxidase-like 2 depletion is protective in age-associated vascular stiffening. Am J Physiol Heart Circ Physiol. 2019;317:H49–H59 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Wheeler JB, Mukherjee R, Stroud RE, Jones JA, Ikonomidis JS. Relation of murine thoracic aortic structural and cellular changes with aging to passive and active mechanical properties. J Am Heart Assoc. 2015;4:e001744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Zingg HH. Vasopressin and oxytocin receptors. Baillieres Clin Endocrinol Metab. 1996;10:75–96 [DOI] [PubMed] [Google Scholar]
  • 38.Mehta PK, Griendling KK. Angiotensin ii cell signaling: Physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol. 2007;292:C82–97 [DOI] [PubMed] [Google Scholar]
  • 39.Nemenoff RA. Vasopressin signaling pathways in vascular smooth muscle. Front Biosci. 1998;3:d194–207 [DOI] [PubMed] [Google Scholar]
  • 40.Das S, Vasanthi HR, Parjapath R. Mitomirs keep the heart beating. Adv Exp Med Biol. 2017;982:431–450 [DOI] [PubMed] [Google Scholar]

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