Short Communication: Asymmetric Dimethylarginine Impairs Angiogenic Progenitor Cell Function in Patients With Coronary Artery Disease Through a MicroRNA-21–Dependent Mechanism
Rationale: The endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) is increased in patients with coronary artery disease and may regulate function of circulating angiogenic progenitor cells (APCs) by small regulatory RNAs.
Objectives: To study the role of microRNAs in ADMA-mediated impairment of APCs.
Methods and Results: By using microarray analyses, we established microRNA expression profiles of human APCs. We used ADMA to induce APC dysfunction and found 16 deregulated microRNAs. We focused on miR-21, which was 3-fold upregulated by ADMA treatment. Overexpression of miR-21 in human APCs impaired migratory capacity. To identify regulated miR-21 targets, we used proteome analysis, using difference in-gel electrophoresis followed by mass spectrometric analysis of regulated proteins. We found that transfection of miR-21 precursors significantly repressed superoxide dismutase 2 in APCs, which resulted in increased intracellular reactive oxygen species concentration and impaired nitric oxide bioavailability. MiR-21 further repressed sprouty-2, leading to Erk Map kinase–dependent reactive oxygen species formation and APC migratory defects. Small interference RNA–mediated superoxide dismutase 2 or sprouty-2 reduction also increased reactive oxygen species formation and impaired APC migratory capacity. ADMA-mediated reactive oxygen species formation and APC dysfunction was rescued by miR-21 blockade. APCs from patients with coronary artery disease and high ADMA plasma levels displayed >4-fold elevated miR-21 levels, low superoxide dismutase 2 expression, and impaired migratory capacity, which could be normalized by miR-21 antagonism.
Conclusions: We identified a novel miR-21–dependent mechanism of ADMA-mediated APC dysfunction. MiR-21 antagonism therefore emerges as an interesting strategy to improve dysfunctional APCs in patients with coronary artery disease.
The endogenous nitric oxide synthase (NOS) inhibitor asymmetrical dimethylarginine (ADMA) is a major risk factor in patients with coronary artery disease (CAD).1–3 Functionally, ADMA induces dysfunction of circulating angiogenic progenitor cells (APCs) (early outgrowth endothelial progenitor cells or circulating angiogenic cells)4 and impairs neovascularization5 by direct inhibition of the endothelial NOS.6 NO plays a crucial role in mobilization, differentiation, and function of APCs,7,8 whereas oxidative stress impairs APC function.9,10
MicroRNAs (miRNAs) are a class of highly conserved, noncoding short RNA molecules that regulate a large portion of the genome. MiRNAs play a crucial role in cardiac biology, and miRNA dysregulation is often found in cardiovascular diseases.11 Knockdown of the miRNA-processing enzyme Dicer in endothelial cells reduces the formation of capillary-like structures by profound dysregulation of angiogenesis-related genes,12 and Dicer-deficient mice die from impaired blood vessel formation and vascularization.13 Recently, miRNAs have been shown to be interesting therapeutic targets in cardiovascular disease, including cardiac fibrosis14 and postmyocardial remodeling.15
In this study, we hypothesized that ADMA exerts effects on the miRNA transcriptome in APCs, leading to functional impairment in vitro and in patients with CAD. In addition, we aimed to identify miRNA-regulated targets and to improve APC function by modulation of an miRNA-dependent mechanism.
We used microarray-based miRNA transcriptome analyses14 and a proteome approach16 to identify miRNA-dependent mechanisms underlying ADMA-mediated dysfunction of human APCs. An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
MicroRNA Expression in Human APCs
To study miRNA expression in human APCs (for characterization, see the Online Data Supplement) a microarray-based approach to screen for the expression of 312 different human miRNAs was used. MiRNAs of the let-7 family, miR-21, -16, -191, -223, 23a, and -23b, were most easily detectable (Online Table I and Figure 1a). The miRNA expression profile of APCs showed a high degree of similarity with that of human umbilical vein endothelial cells and human coronary arterial endothelial cells, although miR-223 was enriched in APCs, whereas miR-126 expression was much higher in mature endothelial cells (see Online Table I).
MicroRNA Expression in ADMA-Induced APC Dysfunction
ADMA mediates APC dysfunction at least in part by impairment of NO bioavailability.4 We thus monitored APC function by testing migratory capacity and used ADMA to induce APC dysfunction. ADMA reduced APC migration in a dose-dependent manner (1 to 10 μmol/L, Figure 1b). We used a pathophysiologically relevant concentration of ADMA (1 μmol/L)4 and performed miRNA transcriptome analysis before and 24 hours after addition of ADMA. ADMA treatment resulted in significant changes of the miRNA transcriptome in APCs. Ten miRNAs were significantly increased (Table). Importantly, the relatively high baseline expression level of miR-21 was further 2.7-fold increased by ADMA treatment. Six miRNAs where significantly repressed by ADMA (Table). Regulation of several miRNAs was validated by miRNA-specific real-time RT-PCR (Figure 1c). We focused our further studies on the role of miR-21 in APC function.
ADMA Increases Reactive Oxygen Species (ROS) Formation, Reduces NO Bioavailability, and Impairs APC Migration by miR-21
To identify miR-21 function in APCs, we transfected miR-21 precursors and inhibitors. Transfection efficiency in APCs and mature endothelial cells was high, based on detection of Cy3-labeled miRNA precursors and increase in miR-21 expression, whereas transfection of miR-21 inhibitors significantly lowered miR-21 expression (Figure 2a and 2b and data not shown). MiR-21 overexpression and ADMA treatment resulted in increased ROS concentration (Figure 2c), reduced NO bioavailability (Online Figure I), and impaired migratory capacity of human APCs, whereas miR-21 inhibition blocked such ADMA-mediated effects (Figure 2c and 2d and Online Figures I and II). There were no significant changes of miR-21 modulation on APC apoptosis (Online Figure III).
MiR-21 Regulates Superoxide Dismutase 2 (SOD2) and the Erk Map Kinase Inhibitor Sprouty2 in APCs
To screen for miR-21 targets, we first used a difference in-gel electrophoresis approach followed by tandem mass spectrometry analysis to search for deregulations in the proteome of human umbilical vein endothelial cells (Figure 2e). MiR-21 overexpression led to altered expression (P<0.05) of 51 proteins 24 hours after treatment (Figure 2e and Online Figure IV and Online Table II). The strongest repression was found for superoxide dismutase type II (SOD2), a protein that is involved in oxidative stress defense. We validated the miR-21–mediated decrease in SOD2 protein expression in cultured APCs by Western blotting (Figure 2f). ADMA also reduced SOD2 protein expression in APCs, which could be blocked by cotransfection with anti–miR-21 (Figure 2f). In addition, SOD2 mRNA expression was repressed by ADMA treatment (Online Figure V). Of note, the 3′UTR of SOD2 displays no miR-21 binding site, based on 3 different bioinformatic prediction tools (PicTar, TargetScan, and miRBase), suggesting an indirect mechanism of action. To study the role of SOD2 in APCs, we treated cells with pegylated SOD2, which resulted in reduced ROS formation and prevention of ADMA-mediated and miR-21–mediated ROS increase (Figure 2g) as well as normalization of migratory capacity (Figure 2h). Interestingly, the effects of ADMA and miR-21 were at least in part dependent on the Erk Map kinase signaling pathway based on inhibition experiments with PD98059 (Figure 2g and 2h). In line, ADMA and miR-21 decreased the Erk Map kinase inhibitor Sprouty2 (SPRY2), leading to enhanced ERK1/2 phosphorylation (Figure 3a). We next tested whether small interfering (si)RNA-mediated reduction in SOD2 or SPRY2 expression would mimic the effects of miR-21 (Figure 3b). Of note, SPRY2 reduction by siRNA also resulted in increased ERK1/2 phosphorylation (Figure 3b). Comparable to the effects of miR-21, the reduction of SPRY2 or SOD2 increased ROS formation and impaired APC migratory capacity (Figure 3c and 3d). Impaired migration of SPRY2-deficient APCs could be completely normalized by Erk Map kinase inhibition but only partly by cotreatment with anti-miR-21. The effect of siRNA-mediated SOD2 reduction on APC migration could not be rescued by miR-21 blockade (Figure 3d), suggesting important roles of SPRY2 and SOD2 in miR-21–mediated APC dysfunction.
MiR-21 Activation in APCs From Patients With CAD
To translate the in vitro findings into a clinical scenario, we isolated APCs from patients with angiographically proven CAD as well as control subjects (no CAD, see Online Table III) and determined ADMA plasma levels, miR-21 expression, and APC function. APCs from CAD patients with high ADMA plasma levels showed significantly increased miR-21 expression levels compared with patients with low ADMA levels or control subjects (Figure 3e). MiR-21 expression of isolated APCs significantly correlated with ADMA plasma levels (r=0.57, P<0.01) and inversely with migratory capacity (r=−0.56; P≤0.05). ADMA plasma levels inversely correlated with SOD2 expression in APCs (r=−0.62; P<0.01; Figure 3f) as determined in a subgroup of an independent study population with CAD.4 To study a functional role of increased miR-21 levels in APC dysfunction of CAD patients, we performed further ex vivo studies. We transfected APCs from CAD patients with miR-21 inhibitors or scrambled controls and investigated APC migratory capacity. Blockade of miR-21 by anti-miR-21 but not by scrambled controls normalized APC function of CAD patients (Figure 3g), identifying miR-21 as a potential therapeutic target in dysfunctional APCs.
ADMA inhibits the enzyme activity of NOSs and contributes to CAD.3,6 In this study, we show a further ADMA-mediated mechanism leading to increased oxidative stress and impaired NO bioavailability in APCs, which play a key role in endothelial regeneration and vascular homeostasis.7 We identified miR-21 to be activated by ADMA and to reduce the expression of superoxide dismutase 2, a key enzyme in oxidative stress defense. In addition, miR-21 led to oxidative stress by Erk Map kinase activation caused by inhibition of SPRY2. This resulted in impairment of NO availability and APC dysfunction. The in vitro data could be translated into APCs from patients with CAD and high ADMA plasma levels. Treatment with miR-21 inhibitors rescued APC function in vitro and ex vivo. Recently, a further report found ADMA to result in increased superoxide production in endothelial cells.17
Because APC dysfunction is mechanistically involved in the onset and progression of endothelial dysfunction and coronary artery disease, specific therapies targeting dysfunctional APCs may be of great clinical value. Indeed, previous studies have suggested that miRNAs may serve as valuable therapeutic targets in a variety of diseases including cardiovascular disease.14,15,18,19 For instance, antagonism of miR-21 has been shown to reduce cardiac fibrosis by a direct effect in cardiac fibroblasts14 and to prevent vascular neointima proliferation after carotid damage.20 In this study, we show that miR-21 is also expressed in APCs and that its inhibition reduced ADMA-mediated oxidative stress and improved cellular migratory capacity. MiR-21 thus emerges as an interesting target in various forms of cardiovascular diseases.
Our study has several limitations: we focused only on APCs characterized as described in the Online Data Supplement and thus it remains to be determined whether the miR-21–dependent mechanism also contributes to dysfunction of other subtypes of vascular progenitor cells. Also, there may be additional mechanisms involved in how ADMA leads to APC dysfunction, such as endothelial NOS uncoupling21 or activation of NADPH oxidases.17 There are probably many more direct and indirect targets of miR-21 in APCs that must be uncovered in future studies. However, our data point to a role of miR-21 in regulation of oxidative stress defense in APCs and identify miR-21 as a potential therapeutic target in patients with CAD to improve impaired APC function.
We acknowledge the skilful technical assistance of A. Horn, M. Kümmel, A. Holzmann, R. Ax-Smolarski, A. Just, and J. Remke.
Sources of Funding
This work was supported by a grant of the Deutsche Forschungsgemeinschaft (TH903/7-1 to T.T. and J.B. and TH903/7-2 to T.T.).
T.T. and J.B. have filed patent applications for the diagnostic and therapeutic use of microRNAs in cardiovascular medicine.
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Novelty and Significance
What Is Known?
MicroRNAs are regulatory RNA molecules and key players in vascular differentiation and endothelial function.
Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor and contributes to endothelial dysfunction and cardiovascular death.
MicroRNAs emerge as potential powerful therapeutic targets in cardiovascular disease.
What New Information Does This Article Contribute?
ADMA regulates microRNAs in circulating angiogenic cells.
ADMA-mediated increase of miR-21 triggers oxidative stress and dysfunction in circulating angiogenic cells via repression of the Erk Map kinase inhibitor Sprouty-2 and superoxide dismutase 2.
Targeting miR-21 rescues ADMA-mediated circulating angiogenic cell dysfunction in vitro and improves functional activity of proangiogenic cells isolated from patients with coronary artery disease.
ADMA levels are increased in patients with coronary artery disease and contribute to endothelial and angiogenic progenitor cell dysfunction and cardiovascular mortality. MicroRNAs posttranscriptionally regulate about one third of genes and play a major role in endothelial homeostasis and function. We show microRNAs to be altered in dysfunctional angiogenic progenitor cells (circulating angiogenic cells). ADMA increased miR-21, which resulted in oxidative stress and dysfunction of angiogenic progenitor cells. MiR-21 antagonism normalized the function of patient-derived circulating angiogenic cells and may serve as a valuable target for the development of future microRNA-based therapeutic treatment strategies for cardiovascular disease.
In April 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.2 days.
Original received January 12, 2010; revision received May 5, 2010; accepted May 7, 2010.