Inhibition of MicroRNA-29 Enhances Elastin Levels in Cells Haploinsufficient for Elastin and in Bioengineered Vessels—Brief Report
Objective—The goal of this study was to determine whether antagonizing microRNA (miR)-29 enhances elastin (ELN) levels in cells and tissues lacking ELN.
Methods and Results—miR-29 mimics reduced ELN levels in fibroblasts and smooth muscle cells, whereas miR-29 inhibition increased ELN levels. Antagonism of miR-29 also increased ELN levels in cells from patients haploinsufficient for ELN and in bioengineered human vessels.
Conclusion—miR-29 antagonism may promote increased ELN levels during conditions of ELN deficiencies.
Elastin (ELN) is a major component of the extracellular matrix in arteries that is essential for normal structural integrity and function. In humans, a single ELN gene is found on chromosome 7, and mutations in 1 allele can lead to several elastinopathies, including supravalvular aortic stenosis (SVAS) and a congenital narrowing of the ascending aorta or other vessels.1 SVAS is also a prominent component of Williams-Beuren syndrome (WBS), a frequent heterozygous deletion of a ≈1.5-Mb segment at chromosome 7q11.23 that includes ELN.2 Despite having 1 intact allele, the expression of ELN is less than 50% of normal in WBS, thus suggesting posttranscriptional modulation of the mRNA.3
See accompanying article on page 548
ELN is secreted into the extracellular space as soluble tropoelastin, which is cross-linked by lysyl oxidase and lysyl oxidase-like 1 to form insoluble, mature ELN fibrils.4 Rodent studies have shown that elastogenesis begins during midgestation, peaks in the perinatal period, and drops sharply thereafter to low levels that persist into adulthood.1 Interestingly, tropoelastin premRNA levels remain elevated in adult rat lungs despite considerably reduced steady-state mRNA levels, thus suggesting that mRNA posttranscriptional regulation may be a predominant mechanism to regulate ELN mRNA and protein levels in adults.5
In the past decade, microRNAs (miRNAs) have emerged as important regulators of gene expression robustness.6 miRNAs predominantly target the 3′-untranslated region of mRNAs, either destabilizing the mRNA transcript or interfering with its translation into protein. Previous work has shown that microRNA (miR)-29 mimics downregulate the expression of ELN, COL1A1, and COL3A1.7 Here, we show that inhibition of miR-29a can dramatically increase ELN expression in human cells and miR-29 inhibition upregulates ELN levels in cells from patients with ELN haploinsufficiencies and in bioengineered human blood vessels. Thus, antagonizing the actions of miR-29 may promote increased ELN levels during conditions of enhanced elastinolysis or deficiencies.
Materials and Methods
Cell Culture and miRNA Treatment
Human dermal fibroblasts, isolated from human foreskin or adult skin, were purchased from Coriell. WBS and age-matched normal human dermal fibroblasts were purchased from the National Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders (University of Maryland, Baltimore, MD). Human vascular smooth muscle (VSM) cells and SVAS pulmonary VSM cell were explanted by outgrowth as described.8 Cells were treated with miRIDIAN miR-29 mimic, hairpin inhibitors or corresponding controls (Dharmacon, Chicago, IL) at 60 nmol/L for 15 hours unless specified otherwise and then were grown in complete media for 48 hours before harvest for RNA or protein studies.
To measure tropoelastin expression in the media, cells were grown in 0.5% FBS Dulbecco's modified Eagle's medium for the final 24 hours and media proteins were precipitated by 10% trichloroacetic acid and resolved on 10% SDS-PAGE gels. To measure ELN protein associated with cell membrane, equal number of cells were lysed in 6 mol/L urea-containing sample loading buffer and loaded onto 10% SDS-PAGE. Western membranes were probed with a polyclonal antibody recognizing human ELN. Ponceau red staining (for media proteins) or β-actin levels (in cell lysates) were used to normalize protein loading.
Culture and Analyses of Tissue Engineered Vessels
Desmosine and Hydroxyproline Analysis
Desmosine and hydroxyproline, demonstrating cross-linked ELN and collagen respectively, were measured as described.11
Results and Discussion
Human dermal fibroblasts were transfected with miRNA mimics for miR-29a, -29b, and -29c or inhibitors (60 nmol/L of each) for 48 hours, and the levels of ELN mRNA were quantified using quantitative reverse transcription–polymerase chain reaction. Figure 1A shows that miR-29a, -29b, and -29c mimics significantly reduced ELN mRNA levels (to 2%–3% of control), whereas transfection with miR-29 inhibitors increased ELN mRNA levels (4–6-fold). Because of homology within the seed sequence of miR-29 family members,7 miR-29 inhibitors also reduced the levels of other miR-29 miRNAs (Figure I in the online-only Data Supplement). Computational analyses have predicted 3 seed sequences (38–44, 297–303, 310–316) for miR-29 binding in the 3′-untranslated region of the human ELN mRNA and these sequences are highly conserved among mammals. As seen in Figure 1B, treatment of human dermal fibroblasts with mimics reduced tropoelastin protein levels, whereas miR-29a, -29b, and -29c inhibitors increased levels compared with controls. To further test whether the miR-29 inhibitor increases major ELN isoforms, we used exon-specific primers to detect the existence of major ELN exons. As seen in Figure 1C, ELN mRNAs containing the known exons 3, 10, 13, 23, and 25 were upregulated to a similar degree with the miR-29a inhibitor.
Because all miR-29 members are abundantly present in the cell types in this study and can potentially regulate ELN levels, we performed additional experiments using only miR-29a. To test the specificity of the miR-29a inhibitor, it was titrated (0–100 nmol/L) in the presence or absence of 60 nmol/L of the miR-29a mimic. As seen in Figure 1D, miR-29a inhibitor (29a-I) dose dependently increases ELN mRNA levels. In the presence of miR-29a mimic (60 nmol/L), more miR-29a inhibitor was needed to achieve the same fold increase of ELN mRNA, but this difference disappeared between 30 and 100 nmol/L of the inhibitor. These data together with data in Figure 1A suggested that (1) the miR-29a inhibitor specifically antagonizes endogenous miR-29, as well as the miR-29a mimic, in a dose-dependent manner, and (2) the miR-29a inhibitor specifically protects ELN mRNA from endogenous miR-29a dependent decay.
Computer analyses also predict several other genes with miR-29 seed sequences, including COL1A1, COL3A1, and VEGF. Overexpression of miR-29 can decrease the activity of luciferase reporter plasmids containing 3′-untranslated regions of COL1A1, COL3A1, and ELN1, and inhibiting miR-29 in vivo can modestly upregulate COL1A1 and COL3A1 mRNA levels in the heart.7 Indeed, miR-29a mimic (60 nmol/L) reduced ELN, COL1A1, COL3A1, and VEGF-A (not shown) mRNA levels in human VSM (Figure 1E). In contrast, the miR-29a inhibitor enhanced ELN expression but did not significantly change the levels of COL1A or COL3A. Additional genes involved in matrix assembly, fibrillin-1 to -3, emilin1, lysyl oxidase 1, and lysyl oxidase-like 1/2, were also tested in VSM and fibroblasts, and miR-29a-I did not increase their expression levels (Figure II in the online-only Data Supplement). The miR-29a inhibitor increased soluble and cell-associated ELN levels in VSM (Figure 1F, left and right). This suggests that miR mimics may achieve concentrations that destabilize multiple transcripts having seed sequences, whereas miR inhibition may selectively upregulate physiological mRNA partners regulated by the endogenous miRNA levels.
Next, we tested the ability of miR-29a inhibitor to upregulate ELN expression in pulmonary VSM cells isolated from a patient with SVAS. A GTAT insertion in exon 9 of the ELN gene caused a frame shift and a premature stop codon in exon 10. Quantitative polymerase chain reaction experiments using primers recognizing ELN exon 18 showed that SVAS cells had low levels of ELN mRNA (≈10% of normal VSM) but equal amounts of endogenous miR-29a compared with normal VSM (Figure IIIA, left, in the online-only Data Supplement). Transfection of SVAS cells with miR-29a inhibitor upregulated ELN expression 3.5-fold (Figure 2A). The expression of COL1A1 and COL3A1 mRNAs were moderately higher in SVAS compared with normal cells (0.68±0.24 versus 0.23±0.09 for COL1A1; 17.5±2.2 versus 9.6±4.6 for COL3A1), and miR-29a inhibitor (60 nmol/L) increased COL1A1 (1.7-fold) but not COL3A1 mRNA levels. miR-29a inhibition increased tropoelastin levels in SVAS cells dose dependently, and in contrast, miR-29 mimic decreased tropoelastin (Figure 2B).
Next, we examined whether miR-29 regulates ELN in dermal fibroblasts isolated from 2 donors with WBS. Comparative genomic hybridization verified that these cells had a typical ≈1.5-Mb microdeletion at 7q11.23 encoding 25 genes, including ELN (Figure IV in the online-only Data Supplement).2 These ELN-haplodeficient cells expressed only 26% to 36% of ELN mRNA levels but comparable levels of miR-29a (Figure IIIA, right, and IIIB, left, in the online-only Data Supplement) compared with age-matched normal fibroblasts from the same bank. Treatment of WBS cells with miR-29 inhibitor upregulated ELN mRNA levels close to those seen in normal cells (Figure 2C and Figure IIIB, middle, in the online-only Data Supplement). In contrast, the levels of COL1A1 were comparable in WBS cells, and miR-29 inhibition increased COL1A1 levels. Western blot analysis showed ELN protein was increased by 40% in WBS cells 48 hours after miR-29 inhibitor treatment (Figure 2D).
Bioengineered human blood vessels are collagen replete but ELN deficient,12 and this paucity of ELN may compromise their mechanical properties. To examine whether miR-29a inhibition can enhance ELN levels and therefore improve vascular mechanics, polyglycolic acid scaffolds were seeded with early passage human VSM as described,12 and vessels were grown in a bioreactor for 6 weeks in the absence or presence of miR-29a inhibitor. As seen in Figure 2E (left), there was little ELN (via transmission electron microscopy) in the control vessels, whereas miR-29a inhibition increased the appearance of ELN “islands” (Figure 2E, right) and its detection by Western blotting (Figure 2F). This ELN was cross-linked as quantified by desmosine content (Figure 2G), and levels of hydroxyproline were comparable between the vessels (Figure 2H). The number of VSM in the vessels were not different between groups (1434±437 versus 1797±453 4′,6-diamidino-2-phenylindole–positive/mm2 for treated versus control vessels, respectively). Finally, biaxial mechanical tests revealed that miR-29a inhibition increased the distensibility of the bioengineered vessels at low pressures (Figure 2I); this response is similar to that of native vessels and suggests that the ELN conferred some structural benefit to the vessel.
Collectively, these data suggest that antagonizing miR-29 may increase functional ELN levels in conditions of ELN haploinsufficiency or during enhanced elastinolysis secondary to aneurysms, emphysema, or aging.
While this work was in review, a new study documented that anti-miR-29a reduces the extent of aneurysms.13
Comparisons between 2 groups were made by unpaired Student t test. Statistical analyses were performed using Prism 4 software (GraphPad). Probability values were 2-tailed, and values <0.05 were considered statistically significant.
Sources of Funding
This work was supported by grants from the Kiev Foundation (to W.C.S., F.J.G., and G.T.) and National Institutes of Health Grants R01-HL64793, R01-HL61371, R01-HL081190, and HL096670 (to W.C.S.).
This manuscript was sent to Kenneth Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.111.238113/-/DC1.
- Received September 2, 2011.
- Accepted November 2, 2011.
- © 2011 American Heart Association, Inc.
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