miR-130a and Tgfβ Content in Extracellular Vesicles Derived from the Serum of Subjects at High Cardiovascular Risk Predicts their In-Vivo Angiogenic Potential

Abstract

Serum-derived extracellular vesicles (sEV) from healthy donors display in-vivo pro-angiogenic properties. To identify patients that may benefit from autologous sEV administration for pro-angiogenic purposes, sEV angiogenic capability has been evaluated in type 2 diabetic (T2DM) subjects (D), in obese individuals with (OD) and without (O) T2DM, and in subjects with ischemic disease (IC) (9 patients/group). sEV display different angiogenic properties in such cluster of individuals. miRNomic profile and TGFβ content in sEV were evaluated. We found that miR-130a and TGFβ content correlates with sEV in-vitro and in-vivo angiogenic properties, particularly in T2DM patients. Ingenuity Pathway Analysis (IPA) identified a number of genes as among the most significant miR-130a interactors. Gain-of-function experiments recognized homeoboxA5 (HOXA5) as a miR-130a specific target. Finally, ROC curve analyses revealed that sEV ineffectiveness could be predicted (Likelihood Ratio+ (LH+) = 3.3 IC 95% from 2.6 to 3.9) by comparing miR-130a and TGFβ content 'in Series'. We demonstrate that sEV from high cardiovascular risk patients have different angiogenic properties and that miR-130a and TGFβ sEV content predicts 'true ineffective sEVs'. These results provide the rationale for the use of these assays to identify patients that may benefit from autologous sEV administration to boost the angiogenetic process.

Conflict of interest statement

G.C., C.C. and M.F.B. are named as inventors in a related patent application. G.C. is a component of the Scientific Advisory Board of UNICYTE.

Figures

Figure 1

Nanosight sEV characterisation. (A) Representative images of NTA analysis referred to each group of patients. (B) Dot-plot graph representing NTA size distribution with mean size values for healthy, obese, T2DM, obese/T2DM and ischemic subjects. (C) The histogram reports the number of EVs recovered from the serum of each group of patients. *p < 0.05 obese and ischemic patients vs healthy subjects (One-way ANOVA followed by Multiple Comparison Test). (n = 9 patients/group). H = Healthy subjects; D = Diabetic patients; O = Obese patients; OD = Obese/Diabetic patients; IC = Ischemic patients.

Figure 2

In-vitro and in-vivo angiogenesis in response to sEVs. (A) Dot plot graph reporting the in-vitro proangiogenic activity of sEV recovered from each patient. The number corresponds to each patient per group (see Supplementary Table 3). The dotted line defines the cut-off for effective and ineffective sEV. The light colour corresponds to ineffective sEV per each group. (B) Representative images of vessels formed in response to effective and ineffective sEV. The number refers to patient sEV. (n = 3 each group except for OD. The same sample was used in 3 independent experiments). (C)In-vivo quantitative analysis of vessels counted in 10 sections of Matrigel for each experimental condition. Data represent the mean value of untreated (C) (n = 3) and treated mice with: healthy ineffective sEV (i-sEV), healthy effective sEV (e-sEV); T2DM ineffective sEV (D i-sEV), T2DM effective sEV (D e-sEV); obese ineffective sEV (O i-sEV), obese effective sEV (O e-sEV); obese/T2DM ineffective sEV (OD i-sEV), obese/T2DM effective sEV (OD e-sEV); ischemic ineffective sEV (IC i-sEV), ischemic effective sEV (IC e-sEV). *p < 0.05 healthy e-sEV vs. healthy i-sEV; §p < 0.05 T2DMe-sEV vs. T2DM i-sEV; #p < 0.05 obese e-sEV vs. obese i-sEV; °p < 0.05 obese/T2DM e-sEV vs. obese/T2DM i-sEV; +p < 0.05 ischemic e-sEV vs. ischemic i-sEV ischemic; (One-way ANOVA followed by Multiple Comparison Test). (n = 3 each group except for OD. The same sample was used in 3 independent experiments) ECs (red), erythrocytes (yellow) and Matrigel (light blue) staining in Matrigel plugs. (Original magnification: x200; scale bar: 12 µm).

Figure 3

TGFβ sEV content. (A) Data obtained per patient/group are reported. The upper curve refers to sEV TGFβ content, while the lower to the % of the test of potency. The dotted line defines the cut-off for effective and ineffective sEV. The number corresponds to each patient (n = 3 each group, except for OD. The same sample was used in 3 independent experiments). (B) Min to max columns represent TGFβ content in effective and ineffective sEVs from each group. *p < 0.05 healthy e-sEV vs. healthy i-sEV; #p < 0.05 T2DM e-sEV vs. T2DM i-sEV; §p < 0.05 obese e-sEV vs. obese i-sEV; (One-way ANOVA followed by Multiple Comparison Test). (n = 3, except for OD. The same sample was used in 3 independent experiments).

Figure 4

miR expression and pattern analysis of sEV. (A) miRNome miRNA qPCR profiler array analysis on healthy effective (e-sEV) and ineffective (i-sEV). miRNAs were detected by RT-PCR. Results are reported as Log2−(∆Ct) (Normalized Relative Quantities) values. Dark blues circles correspond to e-sEV, while light red corresponds to i-sEV (n = 3/each). In red and blue, miRNAs upregulated and down regulated, respectively (t-test, p value < 0.05, see Supplementary Table 5). (B) miRNA validation in patient sEV. RT-PCR for miR-126, miR-21, miR-296-3p, miR-210, miR-130a, miR-27a, miR-29a and miR-191 was performed in all patients and healthy donors. Results are reported as 40-Ct. The red circle indicates patients with high miR-210 expression. (C) Gene Ontology analysis. Network analysis of pathways positively correlated with the miRNAs indicated above. Data were obtained from DIANA-mirPath analyses. The significant enrichment of the TGFβ-associated pathway was identified. (D) miR-130a distribution, patient-by-patient, including healthy subjects is reported and compared with the % of the test of potency. (E) Network analysis of pathways positively correlated with miR-130a. Data were obtained from DIANA-mirPath analysis. Only pathways including at least 15 genes were selected.

Figure 5

miR-130a integrated interaction networks, target validation and ROC analysis. (A) Network analysis between miR-130a and mRNA targets. Lines represent interactions between genes and miR-130a predicted using the IPA Software; indirect interactions (dotted lines), direct interactions (continuous lines). Squares include TGFβ and TGFBR. Circles include genes involved in angiogenesis (KDR, EPHB6, ROCK1, HOXA5). (B) Validation of miR-130a target(s). Upper panel RT-PCR relative quantification (RQ): miR-130a relative amount in untreated ECs (1), in ECs transfected with miRNA Scramble (Scr) (2), with mimic-miR-130a (3), or treated with sEV from patients D17 (4), D18 (5), D20 (6), D1 (7), D2 (8), and D4 (9) is reported. (*p < 0.05 samples 3, 4, 5, 6 vs. samples 1, 2, 7, 8, 9). Lower panel: KDR, ROCK1, HOXA5 and β-actin expression was evaluated in the above samples. sEV from patients D17, D18, D20 are enriched in miR-130a, while sEV from D1, D2 and D4 carry low miR-130a level. (C) ROC analysis. miR-130a and TGFβ sEV content from all patients and healthy subjects were analysed. Predictive capacity was evaluated for each of the two measures individually. A table reporting the AUC values, standard errors, p-values and threshold values is also reported.