MicroRNAs are necessary for vascular smooth muscle growth, differentiation, and function

Abstract

Objective: Regulation of vascular smooth muscle (VSM) proliferation and contractile differentiation is an important factor in vascular development and subsequent cardiovascular diseases. Recently, microRNAs (miRNAs) have been shown to regulate fundamental cellular processes in a number of cell types, but the integrated role of miRNAs in VSM in blood vessels is unknown. Here, we investigated the role of miRNAs in VSM by deleting the rate-limiting enzyme in miRNA synthesis, Dicer.

Methods and results: Deletion of Dicer in VSM results in late embryonic lethality at embryonic day 16 to 17, associated with extensive internal hemorrhage. The loss of VSM Dicer results in dilated, thin-walled blood vessels caused by a reduction in cellular proliferation. In addition, blood vessels from VSM-deleted Dicer mice exhibited impaired contractility because of a loss of contractile protein markers. We found this effect to be associated with a loss of actin stress fibers and partly rescued by overexpression of microRNA (miR)-145 or myocardin.

Conclusions: Dicer-dependent miRNAs are important for VSM development and function by regulating proliferation and contractile differentiation.

Figure 1. Loss of Dicer in smooth muscle is lethal at E16.5-17.5

Two different breeding strategies were used to produce SMC-Dicer KO (Dicer^Δ/Δ + Cre) mice. The number of live pups of different genotypes is shown in A. A total number of 97 pups were genotyped. Timed mating revealed that SM22a-targeted deletion of Dicer results in embryonic lethality at E16.5 to E17.5 (B) and the embryonic lethality was associated with widespread internal hemorrhaging (C). No major difference in embryonic development was observed between Dicer^fl/wt (abbreviated D^fl/wt) and SMC-Dicer KO before E16.5 (D). qPCR analysis of smooth muscle rich umbilical cords demonstrated a significant decrease in Dicer mRNA and miRNAs at E16.5 (E). Data are mean ± SEM from *p<0.05

Figure 2. The loss of Dicer in VSM causes structural abnormalities

Cross-sections of D^fl/wt (A,B) and SMC-Dicer KO (C,D) dorsal aorta from E15.5 embryos. The white boxed region in panels A and C (12,000x) are depicted in panels B and D under higher magnification (60,000x). Arrows denote elastic lamellae and arrowheads point to dense plaques and myofilament arrays seen most prevalently in SMC of wildtype aorta. lu, lumen.

Figure 3. SM22α-targeted deletion of Dicer causes decreased smooth muscle proliferation and aortic remodeling in vessels of E15.5 and E16.5 embryos

Paraffin embedded sections of D^fl/wt and SMC-Dicer KO embryos were stained with hematoxylin and eosin and medial area of the vessel and wall thickness was calculated from the inner and outer media circumference in three sections from each embryo. Bars indicate 100μm. *p<0.05 (A). Paraffin embedded sections from E14.4 embryos were stained for Ki67 antigen (green), SM-α-actin (red) and DAPI (blue; B). The number of Ki67 positive nuclei within the α-actin positive area was quantified as well as the α-actin staining intensity. In a separate experiment the total number of nuclei in the aortic media was quantified in E15.5–E16.5 embryos.

Figure 4. Umbilical vessels from SMC-Dicer KO embryos exhibit reduced contractile function and reduced expression of markers of contractile differentiation

Umbilical arteries from D^fl/wt and SMC-Dicer KO embryos were mounted in a wire myograph. Contractile force in response to depolarization with 80mM K+ as well as passive tension in calcium free solution was analyzed at increasing lumen circumferences (A). Contractile responses to Calyculin A were also blunted in SMC-Dicer KO vessels (B). Western blot analysis of umbilical cords from D^fl/wt and SMC-Dicer KO E16.5 embryos. Original data are shown from different parts of one nitrocellulose membrane and the signal intensity was quantified and normalized to HSP90 (C). Data are mean ± SEM, *p<0.05.

Figure 5. miR-145 rescues SMC-specific gene and protein expression in Dicer KO SMC

(A) Ctrl and Dicer KO SMCs were transfected for 96h with 60nM negative control (Neg Ctrl), miR-145, miR-21, miR-221 or miR-143 mimics. The expression of SMC specific genes (Acta-2: SM-α-actin, Cnn1: calponin, and Myh11: myosin heavy chain), Kcnmb1: B1-subunit of MaxiK channel, Dicer, and miR-145 was analyzed by qPCR and normalized to control VSM cells. 18S was used as internal control for SMC specific genes and Dicer, 5S was used as internal control for miR-145. (B) SMC protein markers and transcription factors were analyzed by Western blotting in Ctrl and Dicer KO SMC transfected with Neg Ctrl or miR-145 mimics. Original data are shown from different parts of one nitrocellulose membrane and the signal intensity was quantified and normalized to HSP90. Data are mean ± SEM, * p<0.05, # p<0.05 vs. KO (Neg Ctrl).

Figure 6. Decreased stress fiber formation in Dicer KO SMC results in loss of SMC-specific gene expression

(A) Ctrl and Dicer KO (KO) SMC and were transfected with negative control (Neg ctrl) or miR-145 mimic for 96h. Actin filaments (F-actin) and monomeric actin (G-actin) were visualized using rhodamine phalloidine (red) and Alexa 488 DNase1 (green), respectively. The staining intensity of F- and G-acting in 12-16 fields from 4 separate slides was quantified and shown as the F/G-actin ratio. (B) Ctrl and Dicer KO SMC were pretreated with vehicle or Latrunculin B (Latr B, 250nM) in order to depolymerize actin filaments. The cells were then transfected with negative control or miR-145 mimic for 24h. The expression of SMC specific genes (Acta-2: SM-a-actin and Myh11: myosin heavy chain) and Dicer was analyzed by qPCR and normalized to Ctrl SMC. 18S was used as internal control. Data are mean ± SEM, * p<0.05