MicroRNA-532-5p Regulates Pericyte Function by Targeting the Transcription Regulator BACH1 and Angiopoietin-1

Figure 1. Effect of Hypoxia on miR Profile in Human APCs (A) Validation of miR532-5p expression by qPCR confirms reduction of intracellular levels following APCs’ exposure to 48 hr hypoxia (i, n = 6 biological replicates) or 48 hr hypoxia followed by return to 48 hr normoxia (ii, n = 4 biological replicates). *p < 0.05 and ***p < 0.001 versus normoxia. (B) MiR-532-5p expression is also affected by cell density (n = 4 biological replicates). *p < 0.05 versus confluent APCs. Values are means ± SE.

Figure 2. miR-532-5p Levels in Human MPCs (A) Effect of 48 hr normoxia or hypoxia (2% O2) on miR-532-5p levels in human MPCs (n = 4 biological replicates). (B) Levels of miR-532-5p in MPCs harvested from healthy (CONTR) or ischemic muscles (CLI) with or without superimposed diabetes (T2D) (n = 5, 3, and 5, respectively). *p < 0.05 versus CONTR, ++p < 0.01 versus CLI. Values are means ± SE.

Figure 3. miR-532-5p Levels in Murine MPCs (A) Bar graph showing the time course of miR-532-5p expression in murine NG2+ MPCs freshly sorted from muscles of mice subjected to unilateral limb ischemia. The red line graph illustrates the corresponding time course of laser Doppler blood flow measurements, which shows a trend opposite to that of miR-532-5p (n = 4 biological replicates, **p < 0.01 and ***p < 0.001 versus time 0). (B) (i) Bar graph showing the miR-532-5p expression levels in NG2+ MCPs freshly sorted from ischemic and contralateral muscles of healthy or streptozotocin-induced type 1 diabetic mice (STZ-T1D) at 3 days from induction of limb ischemia. The red circles represent the area under the curve of laser Doppler blood flow measurements expressed as arbitrary units (AU; n = 4 biological replicates, **p < 0.01 versus MPCs isolated from contralateral non-ischemic muscle). (ii) Bar graph showing capillary density in contralateral muscle 15 days post-ischemia (n = 4, ***p < 0.001 versus control. Values are means ± SE. (iii) Representative images of immunohistological analysis of capillary density. Green staining, isolectin B4; blue staining, DAPI. Scale bar, 50 μm.

Figure 4. Effect of Forced Modulation of miR-532-5p Expression (A) Confirmation of effective transfection by miR modulators, with anti-miR causing miR inhibition (i) and miR mimic causing increased miR expression intracellularly and in conditioned medium (ii and iii); n = 8 biological replicates. *p < 0.05, **p < 0.01, ****p < 0.0001 versus scrambled. (B) Inhibition of miR-532-5p does not affect APC functions. (C) Forced expression of miR-532-5p induces a decrease in metabolic activity and apoptosis; n = 6 biological replicates, *p < 0.05 versus scrambled. (D and E) The effect of miR-532-5p modulation on network formation was investigated in a Matrigel assay. (D) (i) Co-culture of miR-532-5p-inhibited APCs with HUVECs significantly decreased the ability of the latter to form networks on Matrigel compared to scrambled-treated APCs; n = 9 biological replicates, **p < 0.01 versus HUVECs alone, ++p < 0.01 versus scrambled-APCs. (ii) DiL (red) staining of APCs shows miR-532-5p inhibition resulted in no change in the number of APCs found on the network branches or in the nodes (representative images, magnification ×10). (E) (i) Overexpression of miR-532-5p produced a significant increase in…

Figure 5. The Ang-1-Tie-2 Axis Is Involved in the Proangiogenic Effect of miR-532-5p (A) Effects of miR-532-5p inhibition on ANG-1 mRNA (i) and protein levels (ii) in human APCs. (B) Effects of miR-532-5p forced expression on ANG-1 mRNA (i) and protein levels (ii); n = 6 biological replicates per group. *p < 0.05 and **p < 0.01 versus Scrambled. (C) (i) Bar graph showing the effect of Tie-2 inhibition on the inductive effect of miR-532-5p-overexpressing APC conditioned media (CM) on HUVECs network formation. (ii) Representative Matrigel images, ×10 magnification; n = 3 biological replicates per group. *p < 0.01 versus Scrambled-transfected APC CM. Values are means ± SE.

Figure 6. miR-532-5p Regulates ANG-1 via Inhibition of the Transcription Factor BACH1 (A) qPCR (i and ii) and western blotting (iii and iv) demonstrating the effect of miR-532-5p inhibition (i and iii) (n = 4 biological replicates) and overexpression (ii and iv) (n = 5 biological replicates) on BACH1 gene and protein expression in APCs. *p < 0.05 and **p < 0.01 versus scrambled. v, Luciferase activity at 48 hr post-co-transfection of HEK293T cells with miR-532-5p mimics and the following plasmids: pMIR reported (empty vector), 3′ UTR-BACH1, and 3′ UTR-BACH1mut (with mutation of the putative miRNA target site) (n = 5 replicates). ***p < 0.001 versus control, +p < 0.05 versus mimic 3′ UTR BACH1. (B) qPCR, n = 4 (i), and representative western blot image, n = 3 (ii), demonstrating siRNA silenced both BACH1 gene (****p < 0.0001 versus control) and protein expression, respectively. Silencing BACH1 resulted in an increase ANG-1 mRNA (n = 6) (iii) and Ang-1 protein (n = 5) (iv). *p < 0.05 and **p < 0.01 versus scrambled. (C) Inhibition of BACH1 expression produced a significant increase in network formation compared with control (i); representative images, red color: DiL staining of pericytes,…

Figure 7. In Vivo Matrigel Plug Assay Shows miR-532-p Expression Modifies the APC Ability to Stimulate Angiogenesis In Vivo Control, miRNA mimic, or anti-miRNA treated pericytes (GFP-positive) plus HUVECs embedded into Matrigel were implanted subcutaneously into nude mice. (i) Confocal analysis of representative cryostat sections of the Matrigel plug: PCs are stained with an anti-GFP antibody (green) and HUVECs with VE-cadherin antibody (red). Scale bars, 50 μm for large images and 25 μm for the inset; (5 × 5 tiled image). (ii) Results of quantification of areas of endothelium VE-cadherin positive (in percentage in one microscopic view); (iii) ratio of the pericyte-covered endothelium (in percentage). Bars in the graphs represent mean values ± SE, (n = 5) *p < 0.05.