Upregulation of KLF4 in miR-29 null lungs.

<p><b>(A&B)</b> Double IF staining of KLF4 and α-SMA; reduced vasculature α-SMA staining (green) is associated with increased KLF4 staining in the nucleus of cells associated with vessel walls (white arrowheads in <b>D</b>). <b>(C&D)</b> are enlarged images of highlighted areas of <b>A&B</b>. Images are representative of four littermate-matched WT/DKO pairs. <b>(E)</b> Levels of Klf4 mRNA in lungs of wild type and DKO mice (n = 3). <b>(F)</b> Western blot of KLF4 of whole lung protein extracts from three pairs of littermate-matched WT and DKO mice. <b>(G)</b> Densitometric analyses of the blot are presented as relative ratios of KLF4 /GAPDH. Ratio of WT lungs is arbitrarily presented as 1. Data are mean ± SEM; Student’s <i>t</i> test, ** P<0.01.</p

Disruption of miR-29 Leads to Aberrant Differentiation of Smooth Muscle Cells Selectively Associated with Distal Lung Vasculature

<div><p>Differentiation of lung vascular smooth muscle cells (vSMCs) is tightly regulated during development or in response to challenges in a vessel specific manner. Aberrant vSMCs specifically associated with distal pulmonary arteries have been implicated in the pathogenesis of respiratory diseases, such as pulmonary arterial hypertension (PAH), a progressive and fatal disease, with no effective treatment. Therefore, it is highly relevant to understand the underlying mechanisms of lung vSMC differentiation. miRNAs are known to play critical roles in vSMC maturation and function of systemic vessels; however, little is known regarding the role of miRNAs in lung vSMCs. Here, we report that miR-29 family members are the most abundant miRNAs in adult mouse lungs. Moreover, high levels of miR-29 expression are selectively associated with vSMCs of distal vessels in both mouse and human lungs. Furthermore, we have shown that disruption of miR-29 <i>in vivo</i> leads to immature/synthetic vSMC phenotype specifically associated with distal lung vasculature, at least partially due to the derepression of KLF4, components of the PDGF pathway and ECM-related genes associated with synthetic phenotype. Moreover, we found that expression of FBXO32 in vSMCs is significantly upregulated in the distal vasculature of miR-29 null lungs. This indicates a potential important role of miR-29 in smooth muscle cell function by regulating FBXO32 and SMC protein degradation. These results are strongly supported by findings of a cell autonomous role of endogenous miR-29 in promoting SMC differentiation <i>in vitro</i>. Together, our findings suggested a vessel specific role of miR-29 in vSMC differentiation and function by targeting several key negative regulators.</p></div

Upregulation of Foxo3a and Fbxo32 in both miR-29 knockdown cells and miR-29 null lungs.

<p><b>(A)</b> Affymetrix array data of Foxo3a and Fbxo32 in miR-29 knockdown cells (n = 3). <b>(B)</b> qRT-PCR of Foxo3a and Fbxo32 mRNAs in lungs of wild type and DKO mice (n = 3). <b>(C&D)</b> Double IF staining of FBXO32 (red) and α-SMA (green) in WT control and miR-29 DKO lungs. <b>(E&F)</b> are enlarged images of areas highlighted by white dashed lines of <b>C</b> and <b>D</b>. Reduced vasculature α-SMA staining (green) is associated with increased FBXO32 staining (white arrowheads) in cells associated with vessel walls in DKO lungs (<b>D&F</b>). Images are representative of four littermate-matched WT/DKO pairs. Data are mean ± SEM; Student’s <i>t</i> test, * P<0.05.</p

miR-29 promotes the expression of contractile SMC markers.

<p><b>(A)</b> qRT-PCR results of α-SMA and CNN1 mRNAs in PASMCs in which the level of miR-29 is either elevated by miR-29 mimic or knocked down by miR-29 antisense LNA oligos (n = 3); <b>(B)</b> mRNA levels of contractile SMC markers in IMR-90 cells, in which miR-29 is knocked down (n = 3, Affymetrix array data); <b>(C)</b> Levels of α-SMA protein in IMR-90 cells, in which endogenous miR-29 is knocked down. Densitometric analyses of the blot are presented as relative ratios of α-SMA /GAPDH. Ratio of the control is arbitrarily presented as 1. Fold changes of ratio are indicated beneath the corresponding bands. Data are mean ± SEM; Student’s <i>t</i> test, * P<0.05; ** P<0.01.</p

Expression of miR-29 in human lung vasculature.

<p><b>(A, B&C)</b> High miR-29c ISH signal (purple) co-localizes with α-SMA IF staining signal (red) of small vessels (black arrow), while the level of miR-29c in vSMCs (media layer) of nearby large pulmonary arteries (highlighted area by dashed lines) is much lower. <b>(D, E&F)</b> miR-29 is also highly expressed in vSMCs of distal pulmonary artery, where it co-localizes with α-SMA IF staining signal (black arrow). Scale bar: 50μM.</p

Upregulation of COL1A1 in distal vasculature of miR-29 DKO lungs.

<p><b>(A&B)</b> Increased COL1A1 IHC signal is prominently associated with vessel walls, where endogenous miR-29 expression is highly expressed. <b>(C&D)</b> are enlarged images of highlighted areas of <b>A&B</b> by dashed line. All images are representative of four pairs of littermate-matched WT/DKO mice.</p

Disruption of miR-29 expression <i>in vivo</i> leads to aberrant vSMC differentiation.

<p><b>(A&B)</b> Double IF staining of α-SMA and COL1A1; significantly reduced α-SMA staining (green) in distal vasculature was observed in miR-29 null lungs (DKO) as compared to wild type littermate controls (WT). This reduced expression of α-SMA is associated with upregulation of COL1A1(red), a known direct target of miR-29. Images are representative of four pairs of littermate-matched WT/DKO. <b>(C)</b> Immunoblotting analysis of α-SMA, MYOCD, TAGLN and MYH11 of whole lung protein extracts of three littermate-matched WT/DKO pairs. <b>(D)</b> Densitometric analyses of the blots are presented as relative ratios of specific protein/GAPDH. Ratio of the WT control is arbitrarily presented as 1. Data are mean ±SEM from 3 experiments.</p

Postnatal growth retardation and lethality of miR-29 null mice.

<p><b>(A)</b> Expression of miR-29a/b/c is undetectable in lungs of miR-29 DKO mice (n = 3). <b>(B)</b> miR-29 null mice are significantly smaller as compared to their littermates two weeks after birth (n = 5). <b>(C)</b> Representative image of miR-29 null mice at age four weeks. <b>(D)</b> Survival cure of miR-29 null mice (n = 15 for DKO and n = 19 for WT). All miR-29 null mice die within 6 weeks of birth, while none of wild type littermates died in the same period. Data are mean ± SEM; Student’s <i>t</i> test, * P<0.05; ** P<0.01.</p

Expression of miR-29 in mouse lungs.

<p><b>(A)</b> miR-29 family members are the most abundant miRNAs in adult mouse lungs, representing about 19% of total reads of known miRNAs (Next Generation of Sequencing). <b>(B)</b> High levels of miR-29 ISH signal (purple, miR-29c probe, miR-29a or miR-29b probes revealed the same pattern) are associated with vasculature (green arrows), miR-29 in SMCs underneath airway epithelium is much lower (red arrow). <b>(C)</b> Enlarged image of outlined area of <b>B</b>. <b>(D)</b> miR-29 ISH signal (purple) co-localizes with α-SMA IF signal (red) in vessel walls of distal lungs. <b>(E)</b> High levels of miR-29 (purple) in α-SMA positive cells (green) of vessel walls of E18.5 lungs. <b>(F)</b> Levels of miR-29a/b/c in isolated SMCs or endothelial cells (CD31 positive) of adult lungs are significantly higher than those of CD34 positive or Type I epithelial cells (T1α-GFP positive)(P<0.05, Student’s <i>t</i> test). Relative levels were calculated by comparing with levels of miR-29a/b/c in CD34 positive cells (n = 3); Data are mean ± SEM.</p

Upregulation of components of PDGF pathway in both miR-29 knockdown cells and miR-29 null lungs.

<p><b>(A)</b> Affymetrix array data of components of PDGF signaling pathway in miR-29 knockdown cells (n = 3). <b>(B)</b> qRT-PCR of Pdgfrb mRNA in lungs of wild type and DKO mice (n = 3). <b>(C&D)</b> Double IF staining of PDGFRB (red) and α-SMA (green) in WT control and miR-29 DKO lungs. <b>(E&F)</b> are enlarged images of areas highlighted by white dashed lines of <b>C</b> and <b>D</b>. Reduced vasculature α-SMA staining (green) is associated with increased PDGFRB staining (white arrowheads) in cells associated with vessel walls in DKO lungs (<b>D&F</b>). Images are representative of four littermate-matched WT/DKO pairs. Data are mean ± SEM; Student’s <i>t</i> test, * P<0.05.</p