Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition

Abstract

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF₁₆₅, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.

Fig. 1

Preparation of VEGF-MPs and biological characterization in OEPCs. a Schematic representation of the protocol for the preparation of VEGF-conjugated particles and b for the formation of cell aggregates containing sVEGF or VEGF-MPs. c Light microscopy images of the OEPC aggregates at 24 h. The differences in color of cell aggregates are due to the presence of MPs. B ar corresponds to 50 μm. d VEGFR-2 phosphorylation in OEPC aggregates cultured in media containing sVEGF. e VEGFR-2 phosphorylation in OEPC aggregates containing VEGF-MPs or containing cell culture media exposed to the same number of MPs used to make the cell aggregates [(VEGF-MPs)_SN]. VEGF phosphorylation was quantified by ELISA. Values are given as average ± SEM (n = 4–8). f, g ELISA quantification of phospho-Akt/total Akt (f) and phospho-p38/total p38 (g). Values are given as average ± SEM (n = 3). In d, statistical analyses were performed using one-way ANOVA followed by a Bonferroni post test. In e–g, unpaired t-test was performed between groups. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001

Fig. 2

The biological effect of VEGF-MPs on OEPCs. a Single cell calcium measurements. OEPCs were starved in medium without serum for 20 h, loaded with a Ca²⁺ probe and activated by VEGF, blank MPs, VEGF-MPs, or inhibited by Vatalanib, an inhibitor of VEGFR-2. The arrows indicate the time when the compounds or MPs were added. At least 10 cells have been monitored for intracellular Ca²⁺ in each of the experimental groups. Averages and SEM values are in black and grey, respectively. b Schematic representation of the protocols used to demonstrate the higher OEPC survival and activity after exposure to VEGF-MPs than sVEGF. c, d The survival (c 24 h) and apoptosis (d 12 and 24 h) of OEPCs in aggregates under hypoxia in serum-deprived conditions as assessed by an ATP-based assay or the measurement of caspase 9 activity. e, f OEPC aggregates were cultured on Matrigel under hypoxia for 12 and 60 h, after which the tube length (e) and branching points (f) were measured. MPs indicate cell aggregates containing uncoated beads while (VEGF-MPs)_SN indicates cell aggregates exposed to the supernatant of VEGF-MPs. In all graphs, values are given as average ± SEM (n = 3–8). Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001

Fig. 3

OEPC aggregates containing VEGF-MPs improve in vivo survival. OEPC aggregates were prepared with sVEGF, blank MPs or VEGF-MPs and implanted subcutaneously. a Representative IVIS images and b fluorescence intensity measurements of mice following injection of cell aggregates containing 1 million GFP-labeled OEPCs with sVEGF, blank MPs, or VEGF-MPs. The fluorescence intensities were normalized by day one. Results are average ± SEM (n = 7)

Fig. 4

Identification of a miRNA associated with the function of OEPCs after contact with VEGF-MPs. a Schematic representation of the protocol used to identify miRNAs mediating the effect of VEGF-MPs. b Differentially regulated miRNAs (P < 0.05) in OEPC aggregates cultured in vitro for 2 h as evaluated by miRNA array. c Validation of some miRNAs by qRT-PCR. d miRNA expression as evaluated by qRT-PCR in OEPC aggregates implanted subcutaneously in mice for 1 day. U6 was used to normalize the data. In all graphs, values are given as average ± SEM (n = 3–4). Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post test. *P ≤ 0.05,**P ≤ 0.01,***P ≤ 0.001, and ****P ≤ 0.0001. e, g Survival of OEPCs (e) or HUVECs (g) transfected with control antagomiR (Ctrl amiR) or antagomiR-17 (amiR-17), in serum-deprived conditions for 48 h under hypoxia conditions (0.1% O₂), as assessed by Presto-Blue assay. f, h Transfected OEPCs or HUVECs with Ctrl amiR or amiR-17 were cultured on Matrigel for 48 h under hypoxia after which the tube length and branching points were measured. In all graphs, values are given as average ± SEM (n = 4). An unpaired t-test was performed for statistical analysis between Ctrl amiR and amir-17 groups. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001

Fig. 5

AntagomiR-17-treated OEPCs increase blood flow recovery and capillary density in a mouse model of hindlimb ischemia. a, b Unilateral limb ischemia was induced in female CD1 nude mice by occlusion of the left femoral artery. Immediately after occlusion, the ischemic muscles were injected with 3 million antagomiR-17-transfected OEPCs or 3 million control antagomiR-transfected OEPCs. Control group received only EBM-2. Blood flow recovery was measured using high-resolution laser color Doppler imaging and calculated (n = 12 mice/experimental group) as a ratio of ischemic over contralateral foot blood flow. c–e 21 days after surgery, limb muscles were harvested and prepared for immunohistochemical analyses. Capillary density in the adductor muscle was measured by staining with isolectin B4 (revealing endothelial cells) (c). The relative amount of positive cells was counted in eight randomly selected high-power fields (magnification ×20). Scale bars, 20 μm. (d, e). Data were shown as mean ± SEM. One-way ANOVA followed by a Bonferroni post test was used for statistical analysis. *P ≤ 0.05,**P ≤ 0.01, and ***P ≤ 0.001

Fig. 6

Gene targets of miR-17 in OEPCs. a, b mRNA sequencing was performed for OEPCs transfected with control antagomiR or antagomiR-17 for 48 h. a The heat map diagram showing the result of the two-way hierarchical clustering of RNA transcripts and samples, by including the top 500 transcripts (genes) that have the largest log2 fold difference based on FPKM counts. Each row represents one RNA transcript and each column represents one sample. The color of each point represents the relative expression level of a transcript across all samples. The color scale is shown at the bottom: red represents an expression level above the mean; green represents an expression level below the mean. On heat map, C is control amiR while A is amiR-17. b Validation of three gene targets by qRT-PCR. c The expression of previously reported miR-17 gene targets in HUVECs and OEPCs transfected with amiR-17. The results were normalized to control amiR group for each gene. Among the genes tested, CDKN1A was significantly upregulated in both of the cell types transfected with amiR-17. The upregulation in the expression of CDKN1A was also confirmed in OEPC aggregates containing conjugated VEGF in both 24 h in vitro (d) and 24 h in vivo (e) samples. The gene expression results were normalized to cell control group (cell aggregates). Results are average ± SEM (n = 4–8). In b and c, unpaired t-test was performed between ctrl amiR and amir-17 groups, while one-way ANOVA followed by a Bonferroni post test was used among groups in d and e for statistical analysis. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001

Fig. 7

AmiR-17 exerts its pro-survival effect by upregulating CDKN1A. a HEK293 cells were double transfected with 100 ng of each 3′UTR luciferase reporter vectors (empty vector, CDKN1A vector or ZNF652 vector) and 50 nM miRNA mimics (control miRNA or miR-17). Signals from 3′UTR reporters for the CDKN1A and ZNF652 were significantly knocked down when co-transfected with miR-17, but not with control miRNA. No difference between control miRNA and miR-17 was observed when the empty vector was used. b, c OEPCs cultured in monoculture were silenced for ZNF652 and CDKN1A by the use of siRNA. After 2 days of transfection, OEPCs were washed and the cell culture medium was replaced by EBM-2 and cells were incubated under hypoxia conditions (0.1% O₂), with 5% CO₂. After 48 h, both cell survival (by a Presto-Blue cell viability assay (b)) and cell apoptosis (c) were evaluated. In b and c, the values are given as average ± SEM (n = 10). Statistical analyses were performed using unpaired t-test. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001. d Schematic representation of the action mechanism of the VEGF-conjugated microparticles used in this study. The interaction of conjugated VEGF with VEGFR-2 prolongs the phosphorylation of the receptor, calcium signaling and Akt phosphorylation compared with soluble VEGF group. Conjugated VEGF also downregulates miR-17, which leads to the upregulation of CDKN1A expression. Activation of Akt and downregulation of miR-17 lead to an increase in cell survival by reducing apoptosis and favor sprout formation