MicroRNA-377 regulates mesenchymal stem cell-induced angiogenesis in ischemic hearts by targeting VEGF

Abstract

MicroRNAs have been appreciated in various cellular functions, including the regulation of angiogenesis. Mesenchymal-stem-cells (MSCs) transplanted to the MI heart improve cardiac function through paracrine-mediated angiogenesis. However, whether microRNAs regulate MSC induced angiogenesis remains to be clarified. Using microRNA microarray analysis, we identified a microRNA expression profile in hypoxia-treated MSCs and observed that among all dysregulated microRNAs, microRNA-377 was decreased the most significantly. We also validated that vascular endothelial growth factor (VEGF) is a target of microRNA-377 using dual-luciferase reporter assay and Western-blotting. Knockdown of endogenous microRNA-377 promoted tube formation in human umbilical vein endothelial cells. We then engineered rat MSCs with lentiviral vectors to either overexpress microRNA-377 (MSC miR-377) or knockdown microRNA-377 (MSC Anti-377) to investigate whether microRNA-377 regulated MSC-induced myocardial angiogenesis, using MSCs infected with lentiviral empty vector to serve as controls (MSC Null). Four weeks after implantation of the microRNA-engineered MSCs into the infarcted rat hearts, the vessel density was significantly increased in MSC Anti-377-hearts, and this was accompanied by reduced fibrosis and improved myocardial function as compared to controls. Adverse effects were observed in MSC miR-377-treated hearts, including reduced vessel density, impaired myocardial function, and increased fibrosis in comparison with MSC Null-group. These findings indicate that hypoxia-responsive microRNA-377 directly targets VEGF in MSCs, and knockdown of endogenous microRNA-377 promotes MSC-induced angiogenesis in the infarcted myocardium. Thus, microRNA-377 may serve as a novel therapeutic target for stem cell-based treatment of ischemic heart disease.

Figure 1. miRNA expression profile is determined in hypoxia-treated mesenchymal stem cells (MSCs).

(A): A heat map of all dys-regulated miRNAs in hypoxia-treated rat MSCs miRNA microarray. All of the miRNA array raw data are available in the online supplemental Table (n = 5 independent experiments). (B): Microarray data are summarized by volcano plot graph, which displays both fold-change and t-test criteria (log odds). MiR-377 and miR-210 are the most significantly dysregulated miRs in hypoxia-treated rat MSCs compared to normoxia-treated MSCs (the green stands for down-regulation, and the red stands for upregulation). (C): Alterations in expression levels of miR-377 was validated by qPCR (normalized to control U6, n = 5, p<0.05).

Figure 2. Suppression of miR-377 induced in vitro formation of capillary-like structures.

(A): qPCR analysis after normalization against U6 verified the knockdown of endogenous miR-377 (Anti-377) and overexpression of miR-377 (miR-377) into HUVECs transfected with either miR-377 inhibitor or mimic. (B): In vitro tube formation assay indicated that knockdown of miR-377 enhanced the formation of capillary-like structures, but this effect was limited by miR-377 overexpression. Scale bars = 500 µm. (C): Total capillary tube lengths and tube branch points were measured by analytical software Image-Pro Plus 6.0 (IPP). All values were expressed as means ± SE; n = 6 independent experiments for each group; *P<0.05.

Figure 3. Dual-luciferase reporter assay validates that miR-377 directly targets VEGF.

(A): Computational miRNA target prediction analysis coincidentally reveals that the fragment 5′-GAAUCAC-3′ of miRNA-377 pairs well with the fragment 5′-GUGAUUC-3′ located at the 1568–1574 nt of VEGF 3′ UTR, which is a highly conserved site (red fonts) in most of mammals (e.g. rat, human, chimpanzee, rhesus, bushbaby, treeshrew, mouse). (B): Schematic diagram of Dual-Luciferase reporter vector (pEZX-MT01) carrying the VEGF3′ UTR. (C): Quantitative data for dual-luciferase reporter assay results. All values were expressed as means ± SE; n = 6 for each group. *P<0.05 was considered statistically significant.

Figure 4. miR-377 directly down regulates the expression of VRGF in MSCs.

(A): qPCR analysis after normalization against β-actin showed that miR-377 mimic (miR-377) downregulated VEGF mRNA in MSCs, while miR-377 inhibitor (Anti-377) upregulated VEGF mRNA in MSCs. MSCs transfected with a scrambled sequence according to miR-377 mimic and miR-377 inhibitor as negative control (NC^miR and NC^Anti respectively). (B): Western blot assay showed protein level changes of VEGF in MSCs induced by miR-377 mimic (miR-377) and miR-377 inhibitor (Anti-377) as well as its quantitative data. All values were expressed as means ± SE; n = 8 for each group; *P<0.05 was considered statistically significant. (C): qPCR analysis after normalization against β-actin showed significant up-regulation of VEGF in hypoxia-treated MSCs in comparison with normoxia-treated MSCs, which was further increased in MSC^Anti-377, when compared with NC group and miR-377 groups. All values were expressed as means ± SE; n = 8 for each group. *P<0.05 was considered statistically significant.

Figure 5. Pro-angiogenic effects elicited by knockdown of miR-377 are largely dependent on VEGF.

In vitro Tube Formation Assay showed that total capillary tube lengths (A) and tube branch points (B) were significantly reduced by the VEGF siRNA transfection in miR-377-knockdown HUVECs. All values were expressed as means ± SE; n = 6 for each group; ^* P<0.05 was considered statistically significant.

Figure 6. Engineering rat MSCs with lentiviral vectors to overexpress or knockdown of miR-377.

(A): Schematic diagram of recombinant lentiviral vectors. Lenti-GFP, lentiviral empty vector; Lenti-miR-377, lentiviral miR-377 overexpressing vector; Lenti-Anti-miR-377, lentiviral miR-377 inhibitor expression vector. (B): Nearly 100% of MSCs were transfected with Lenti-GFP (MSC^Null), Lenti-miR-377 (MSC^miR-377) or Lenti-Off-miR-377 (MSC^Anti-377) after 72-hour lentiviral infection, as indicated by GFP fluorescence. No morphological changes were found among MSC^Null, MSC^miR-377, and MSC^Anti-377. Scale bars = 200 µm. (C): qPCR analysis after normalization against U6 showed that the knockdown of miR-377 in MSC^Anti-377 and the overexpression of miR-377 in MSC^miR-377. (D): qPCR analysis after normalization against β-Actin. (E): Western-blot consistently showed that the expression of VEGF was reduced in MSC^miR-377 while increased in MSC^Anti-377, compared with that in MSC^Null. All values were expressed as means ± SE; n = 6 for each group; ^* P<0.05 vs. MSC^Null; ^# P<0.05 vs. MSC^miR-377.

Figure 7. Knockdown of endogenous miR-377 enhances MSC-mediated myocardial angiogenesis in vivo.

(A): Slice sections of heart samples, collected from MI rats 4 weeks after the injection of miR-engineered MSCs, were triple-stained with troponin I (cTnT) antibody (Ab) (for cardiomyocytes, red), α-smooth muscle actin (SMA) Ab (for vascular cells, green; white arrows) and DAPI (for nuclei, blue). Vascular density was measured in (A) MSC^Null group, (B) MSC^miR-377 group, and (C) MSC^Anti-377group. Capillary density was identified by Von Willebrand (vWF) staining (green; white arrows) in (D) MSC^Null group, (E) MSC^miR-377 group and (F) MSC^Anti-377 group. IPP software was used to quantitatively analyze (G) vascular density, and capillary density in different treatment groups. All values were expressed as means ± SE; n = 6 for each group. ^* P<0.05 vs. MI; ^# P<0.05 vs. MI+MSC^Null.

Figure 8. Injection of miR-377-knockdown MSCs into the rat infarcted hearts limits fibrosis and improves cardiac functions.

(A): 5-µm sections of heart slices, collected from MI rats 4 weeks after the injection of miR-engineered MSCs, were stained with Masson-Trichome. (B): Percentage of fibrosis in left ventricle (LV) in various treatments determined with Image J software. All values were expressed as means ± SE; n = 6 for each group. *P<0.05 was considered statistically significant. (C): The LV end-diastolic diameters (LVDd, yellow arrow) and LV end-systolic diameters (LVDs, yellow arrow) in MSC^Null group, MSC^miR-377 group and MSC^Anti-377 group were measured using echocardiographic M-mode recordings. All echocardiographic measurements were averaged from at least 3 separate cardiac cycles. (D): Quantitative data of LVDd, LVDs, LV ejection fraction (EF) and fractional shortening (FS) were analyzed and compared among various groups. All values were expressed as means ± SE; n = 6 for each group. ^* P<0.05 vs. MI; ^# P<0.05 vs. MI+MSC^Null.