Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31

Abstract

Cell secretion is an important mechanism for stem cell-based therapeutic angiogenesis, along with cell differentiation to vascular endothelial cells or smooth muscle cells. Cell-released microvesicles (MVs) have been recently implicated to play an essential role in intercellular communication. The purpose of this study was to explore the potential effects of stem cell-released MVs in proangiogenic therapy. We observed for the first time that MVs were released from adipose-derived stem cells (ASCs) and were able to increase the migration and tube formation of human umbilical vein endothelial cells (HUVECs). Endothelial differentiation medium (EDM) preconditioning of ASCs upregulated the release of MVs and enhanced the angiogenic effect of the released MVs in vitro. RNA analysis revealed that microRNA was enriched in ASC-released MVs and that the level of microRNA-31 (miR-31) in MVs was notably elevated upon EDM-preconditioning of MV-donor ASCs. Further studies exhibited that miR-31 in MVs contributed to the migration and tube formation of HUVECs, microvessel outgrowth of mouse aortic rings, and vascular formation of mouse Matrigel plugs. Moreover, factor-inhibiting HIF-1, an antiangiogenic gene, was identified as the target of miR-31 in HUVECs. Our findings provide the first evidence that MVs from ASCs, particularly from EDM-preconditioned ASCs, promote angiogenesis and the delivery of miR-31 may contribute the proangiogenic effect.

Significance: This study provides the evidence that microvesicles (MVs) from adipose-derived stem cells (ASCs), particularly from endothelial differentiation medium (EDM)-preconditioned ASCs, promote angiogenesis. An underlying mechanism of the proangiogenesis may be the delivery of microRNA-31 via MVs from ASCs to vascular endothelial cells in which factor-inhibiting HIF-1 is targeted and suppressed. The study findings reveal the role of MVs in mediating ASC-induced angiogenesis and suggest a potential MV-based angiogenic therapy for ischemic diseases.

Keywords: Adipose stem cell; Angiogenesis; Endothelial cell; Microvesicle; miRNA.

Figure 1.

Induction of migration and tube formation of HUVECs by CdM from adipose-derived stem cells (ASCs). ASCs were incubated in endothelial basal medium/1% fetal bovine serum (FBS) for 2 days in the absence (A, B) or presence (C, D) of GW4869, an MV-formation inhibitor. CdM was collected and used to treat HUVECs. Cell migration (A, C) and tube formation (B, D) assays for treated HUVECs were performed. The endothelial basal medium/1% FBS without cells was incubated in parallel and was used as a control. The CdM with removal of MVs via ultracentrifugation was used as CdM-MV free. Representative images of cell migration and tube formation are displayed. Scale bar = 200 µm. (A–D): n = 4. ∗, p < .05 versus CdM; ∗∗, p < .01 versus 0 µM. Abbreviations: CdM, conditioned medium; HUVEC, human umbilical vein endothelial cell; MV, microvesicle.

Figure 2.

Induction of migration and tube formation of HUVECs by MVs from adipose-derived stem cells (ASCs). (A): Conditioned medium was subjected to standard serial centrifugation for MV isolation. The isolated pellet was examined with scanning electron microscopy (scale bar = 1 µm). (B): ASCs were maintained in growth medium or preconditioned with endothelial differentiation medium for 4 days. After washing, the ASCs were then incubated for 2 days in endothelial basal medium/1% FBS with or without the presence of GW4869. Western blot analysis of the MVs was performed with Alix, an MV marker. Each lane represented an MV lysate from 3 × 10⁶ cells. (C): The protein content of MVs and MV-P from ASCs were determined. (D–F): HUVECs were treated with 30 µg/ml (protein concentration) MV or MV-P. HUVECs in fresh endothelial basal medium/1% FBS was used as a control. Cell migration (D), tube formation (E), and proliferation (F) assays of the HUVECs were performed as described in Material and Methods. (C–F): n = 4. ^##, p < .01 versus MV or control; ∗∗, p < .01 versus control. Abbreviations: HUVEC, human umbilical vein endothelial cell; MV, microvesicle; MV-P, microvesicles from adipose-derived stem cells preconditioned with endothelial differentiation medium; OD, optical density.

Figure 3.

Analysis of RNA extracted from ASC-released MVs. (A): Small RNA (fewer than 200 nucleotides) and large RNA extracted from ASCs and ASC-released MVs were quantified (n = 3– 4). Total RNA was set to 100%. (B): Equal amounts of total RNA from ASCs and MVs were analyzed using Bioanalyzer (Agilent Technologies). 18S and 28S indicated the ribosomal RNA. (C): Small RNA from MVs was analyzed using Bioanalyzer. (D): An electropherogram of the lane MV on (C). (E): Reverse-transcriptase-polymerase chain reaction analysis of miRNA for small RNA extracted from MV-P. The level of miRNA in MV was set to 1 (dashed line). U6 was used as an internal control. n = 4. ∗, p < .05; ∗∗, p < .01. Abbreviations: ASC, adipose-derived stem cell; miRNA, microRNA; MV, microvesicle; nt, nucleotide.

Figure 4.

miR-31 contributes to the proangiogenesis induced by MV-P in vitro. (A–C): Adipose-derived stem cells (ASCs) were transduced with lentiviral ZipmiR-31 to silence miR-31. ZipmiR-Cont was used as a control. MVs and MV-P were obtained from the transduced ASCs and were used to treat HUVECs. The miR-31 content in HUVECs (A), and the cell migration (B) and tube formation (C) of HUVECs were determined. (D): HUVECs were transfected with commercial pre-miR-31. A pre-miR-Cont was used as a control. The tube formation of HUVECs was measured 48 hours after transfection. (A–D): n = 4–5. ∗, p < .05; ∗∗, p < .01. Abbreviations: HUVEC, human umbilical vein endothelial cell; miR-31, microRNA-31; MV, microvesicle; MV-P, microvesicles from adipose-derived stem cells preconditioned with endothelial differentiation medium.

Figure 5.

miR-31 contributes to the proangiogenesis induced by MV-P ex vivo and in vivo. Adipose-derived stem cells (ASCs) were transduced with lentiviral ZipmiR-31 to silence miR-31. ASCs untransduced (control) or transduced with a ZipmiR-Cont were used as controls. MV and MV-P were obtained from these ASCs. (A): Mouse aortic rings were collected and treated with various MVs, as indicated, for 5 days (n = 8). Representative images (upper panel) and a statistical analysis of the outgrowth area of aortic rings (lower panel) for each treatment condition are displayed. The outgrowth area of aortic rings treated with MV from untransduced cells was set to 1. Scale bar = 100 µm. (B): PBS, MV, or MV-P was mixed with Matrigel and injected subcutaneously into the flanks of the nude mice. The Matrigel plugs were harvested 2 weeks postimplantation (n = 6). Upper panel: Representative pictures of the plugs are exhibited. Scale bar = 5 mm. Middle panel: The sections of the plugs were subject to immunohistochemistry analysis for CD31, an endothelial cell marker, and counterstained with DAPI. Scale bar = 100 µm. Lower panel: Quantification of the CD31-positive area was performed. The positive area in the slide from the plugs containing PBS was set to 1. ∗, p < .05; ∗∗, p < .01. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; miR-31, microRNA-31; MV, microvesicle; MV-P, microvesicles from adipose-derived stem cells preconditioned with endothelial differentiation medium; PBS, phosphate-buffered saline.

Figure 6.

MV-P suppresses FIH1 expression in human umbilical vein endothelial cells (HUVECs) via the delivery of miR-31. (A): The levels of predicted miR-31 target mRNA in MV or MV-P-treated HUVECs were examined with reverse-transcriptase polymerase chain reaction (RT-PCR). β-actin was used as an internal control. The mRNA levels in MV-treated HUVECs were set to 1.0 (dashed line) (n = 5). (B, C): HUVECs were transfected with pre-miR-Cont (control) or pre-miR-31. The mRNA and protein levels of FIH1 in transfected HUVECs were determined with RT-PCR (n = 3) (B) and Western blot (C), respectively. (D): Schematic representation of the luciferase-reporter lentivirus containing insert of FL or HL of FIH1 3′-UTR. Four predictive target sites by miR-31 are shown. A mutation at the site of 117–124 of FIH1 3′-UTR is illustrated. The seed site of mature miR-31 is highlighted. (E): HUVECs were transfected with pre-miR-Cont or pre-miR-31 followed by transduction of indicated lentiviral reporters the next day. The luciferase activity in HUVECs was determined using luminometry 2 days posttransduction (n = 5). (F): MV and MV-P from ZIPmiR-31-transduced adipose-derived stem cells (ASCs) were used to treat HUVECs. MV and MV-P from untransduced (control) or ZIPmiR-Cont-transduced ASCs were used as controls. The protein levels of FIH1 in treated HUVECs were determined using Western blot. β-actin was used as an internal control. (G): A statistical analysis of the density of the Western blot bands in (F) using Image J software. ∗, p < .05; ∗∗, p < .01. Abbreviations: FL, full length; HL, half length; miR-31, microRNA-31; MV, microvesicle; MV-P, microvesicles from adipose-derived stem cells preconditioned with endothelial differentiation medium; NS, not statistically significant.