Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster
Thomas Boettger, … , Lutz Hein, Thomas Braun
Thomas Boettger, … , Lutz Hein, Thomas Braun
Published August 17, 2009
Citation Information: J Clin Invest. 2009;119(9):2634-2647. https://doi.org/10.1172/JCI38864.
View: Text | PDF
Research Article

Acquisition of the contractile phenotype by murine arterial smooth muscle cells depends on the Mir143/145 gene cluster

Abstract

VSMCs respond to changes in the local environment by adjusting their phenotype from contractile to synthetic, a phenomenon known as phenotypic modulation or switching. Failure of VSMCs to acquire and maintain the contractile phenotype plays a key role in a number of major human diseases, including arteriosclerosis. Although several regulatory circuits that control differentiation of SMCs have been identified, the decisive mechanisms that govern phenotypic modulation remain unknown. Here, we demonstrate that the mouse miR-143/145 cluster, expression of which is confined to SMCs during development, is required for VSMC acquisition of the contractile phenotype. VSMCs from miR-143/145–deficient mice were locked in the synthetic state, which incapacitated their contractile abilities and favored neointimal lesion development. Unbiased high-throughput, quantitative, mass spectrometry–based proteomics using reference mice labeled with stable isotopes allowed identification of miR-143/145 targets; these included angiotensin-converting enzyme (ACE), which might affect both the synthetic phenotype and contractile functions of VSMCs. Pharmacological inhibition of either ACE or the AT1 receptor partially reversed vascular dysfunction and normalized gene expression in miR-143/145–deficient mice. We conclude that manipulation of miR-143/145 expression may offer a new approach for influencing vascular repair and attenuating arteriosclerotic pathogenesis.

Authors

Thomas Boettger, Nadine Beetz, Sawa Kostin, Johanna Schneider, Marcus Krüger, Lutz Hein, Thomas Braun

×

Figure 6

ACE is a target of miR-145.

Options: View larger image (or click on image) Download as PowerPoint
ACE is a target of miR-145. Analysis of protein expression by SILAC mous...
Analysis of protein expression by SILAC mouse–based quantitative proteomics and Western blot analysis. (A and B) SILAC-labeled (13C6Lys) WT aorta was mixed with nonlabeled WT aorta (A) and miR-143/145 KO aorta (B). Protein ratios are plotted against added peptide intensities. Blue dots represent proteins that do not show a significant change in expression levels (P ≥ 0.05). Red, yellow, and green dots represent proteins with a significant change in expression levels at different confidence intervals (red: P < 0.05, yellow: P < 0.01, green: P < 0.001). Circled green dots in A and B indicate fatty acid–binding protein. (C) To obtain ratios between nonlabeled WT and nonlabeled Mir143/145-KO aortae, both ratios from 13C6Lys-WT/WT (nonlabeled) and 13C6Lys-WT/miR-143/145 KO (non-labeled) were divided by each other. (D and E) Mass spectra of an ACE-1 peptide [YVEFSNK (MH2+)] SILAC pair with 13C6Lys-WT/WT (D) and a pair 13C6Lys-WT/Mir143/145-KO (E). Peaks with lower molecular weight correspond to the unlabeled peptide; peaks with higher molecular weight (greater than 6 Da) correspond to the 13C6Lys peptide. Calculation of ACE-1 signals from all samples revealed a 4.9-fold upregulation of ACE-1 in KO vs. WT aortae (n = 3 per genotype, P < 0.01; see Supplemental Table 1). (F and G) Immunofluorescence analysis of ACE expression in cryosections of aorta (scale bar: 25 μm). (H) Western blot analysis of ACE-1 expression in WT and Mir143/145-KO aortae (n = 4 per genotype, P < 0.001, KO/WT ratio: 4.3). (I) miR-145 binding sites in the 3ι UTR and ORF of ACE predicted by the miRanda algorithm. (J) Schematic representation of the role of miR-143/145 in VSMCs.