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Received: 27 February 2017 Accepted: 10 July 2017 Published: xx xx xxxx

Serum-derived extracellular vesicles (EVs) impact on vascular remodeling and prevent muscle damage in acute hind limb ischemia Claudia Cavallari1, Andrea Ranghino1, Marta Tapparo1, Massimo Cedrino1, Federico Figliolini1, Cristina Grange1, Valentina Giannachi1, Paolo Garneri2, Maria Chiara Deregibus1, Federica Collino1, Pietro Rispoli2, Giovanni Camussi1 & Maria Felice Brizzi   1 Serum is an abundant and accessible source of circulating extracellular vesicles (EVs). Serum-EV (sEV) pro-angiogenic capability and mechanisms are herein analyzed using an in vitro assay which predicts sEV angiogenic potential in vivo. Effective sEVs (e-sEVs) also improved vascular remodeling and prevented muscle damage in a mouse model of acute hind limb ischemia. e-sEV angiogenic proteomic and transcriptomic analyses show a positive correlation with matrix-metalloproteinase activation and extracellular matrix organization, cytokine and chemokine signaling pathways, Insulin-like Growth Factor and platelet pathways, and Vascular Endothelial Growth Factor signaling. A discrete gene signature, which highlights differences in e-sEV and ineffective-EV biological activity, was identified using gene ontology (GO) functional analysis. An enrichment of genes associated with the Transforming Growth Factor beta 1 (TGFβ1) signaling cascade is associated with e-sEV administration but not with ineffective-EVs. Chromatin immunoprecipitation analysis on the inhibitor of DNA binding I (ID1) promoter region, and the knock-down of small mother against decapentaplegic (SMAD)1–5 proteins confirmed GO functional analyses. This study demonstrates sEV pro-angiogenic activity, validates a simple, sEV pro-angiogenic assay which predicts their biological activity in vivo, and identifies the TGFβ1 cascade as a relevant mediator. We propose serum as a readily available source of EVs for therapeutic purposes. Critical limb ischemia (CLI), the most severe consequence of peripheral artery disease, is a rife condition caused by acute occlusion of the peripheral arteries1. Although surgical or endovascular intervention are still the standard therapy for improving blood flow2, most patients complain of persistent or recurring symptoms even after successful revascularization3. This implies that new therapeutic options for patients complying these symptoms are very much still a major unmet need. The formation of newly born capillary blood vessels, neovascularization, is a crucial event in rescuing tissue after ischemia. A vast array of pre-clinical and clinical data indicates that stem cells can contribute to the healing processes by improving vascularization4, 5. However, the transient detection of stem cells at the site of tissue damage has suggested that paracrine mechanisms may contribute to stem cell action6, 7. “Exosomes”, vesicles derived from the endosomal membrane compartment by exocytosis, and “ectosomes/microvesicles”, vesicles generated by the budding of cell plasma membranes8 are included among such paracrine mechanisms. Due to the overlap in the characteristics and biological activity of exosomes and ectosomes/microvesicles9, it has recently been suggested that a more inclusive term, “extracellular vesicles” (EVs), be used. EVs are powerful cell-to-cell communication mediators in both physiological and pathological conditions10. Recent studies have provided evidence that the transfer of EV cargo can dictate recipient cell fate11, 12. Indeed, the transfer of EV cargo, including nucleic acids and proteins, into recipient cells is the most relevant mechanism of EV action13, 14. The most studied 1

Department of Medical Sciences, 2i3T Scarl, University of Turin, Turin, Italy. 2Department of Surgical Sciences, University of Turin, Turin, Italy. Claudia Cavallari and Andrea Ranghino contributed equally to this work. Correspondence and requests for materials should be addressed to G.C. (email: [email protected]) or M.F.B. (email: [email protected])

Scientific REPOrTS | 7: 8180 | DOI:10.1038/s41598-017-08250-0

1

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sEVs

Angiogenesis assay %

Proliferation assay %

Average %

Results

1

42.8 ± 1.4

62.5 ± 2.3

52.6 ± 1.9

e-sEV

2

54.4 ± 3.9

56.4 ± 3.1

55.4 ± 3.5

e-sEV

3

31.9 ± 1.4

89.5 ± 0.5

60.7 ± 1.0

e-sEV

4

53.3 ± 0.3

64.1 ± 1.1

58.7 ± 0.7

e-sEV

5

68.7 ± 2.6

55.5 ± 3.1

62.1 ± 2.9

e-sEV

6

43.8 ± 0.9

77.6 ± 4.0

60.7 ± 2.5

e-sEV

7

88.5 ± 4.2

66.2 ± 3.3

77.4 ± 3.8

e-sEV

8

65.5 ± 0.6

44.4 ± 0.5

54.9 ± 0.6

e-sEV

9

56.5 ± 4.5

50.1 ± 3.9

53.3 ± 4.2

e-sEV

10

31.5 ± 1.1

99.8 ± 5.6

65.7 ± 3.4

e-sEV

11

35.2 ± 8.3

8.5 ± 4.1

21.9 ± 6.2

i-sEV

12

99.2 ± 0.5

32.2 ± 3.4

65.7 ± 2.0

e-sEV

13

28.8 ± 3.2

24.5 ± 2.3

26.7 ± 2.8

i-sEV

14

41.7 ± 6.6

68.6 ± 4.5

55.2 ± 5.6

e-sEV

15

50 ± 3.8

55 ± 4.9

52.5 ± 4.4

e-sEV

16

8.3 ± 1.3

19.1 ± 4.0

13.7 ± 2.5

i-sEV

17

66.7 ± 4.5

41.1 ± 4.2

53.9 ± 6.6

e-sEV

18

15.1 ± 1.5

3.6 ± 0.6

9.5 ± 2.6

i-sEV

Table 1. Proangiogenic in vitro effects of different sEV samples. 18 sEV samples evaluated by in vitro angiogenesis assay and in vitro proliferation assay on ECs. The average represents the mean value of results of both assays ± SD (see Methods). sEVs with an average value exceeding 50% have been considered effective sEVs (e-sEVs) otherwise they are ineffective (i-sEVs).

EVs are currently those released by stem cells, thanks to their ability to mimic the effects of the cell of origin14. A range of different phenotypic changes, depending on the pathological setting, have been described in target cells15. Transcriptomic and proteomic analyses have often been performed in an attempt to try to characterize EV mechanism of action11, 16. In particular, a robust pro-angiogenic profile has been described in EVs recovered from mesenchymal stem cells derived from bone marrow or adipose tissues5, 17. These observations have hinted at the potential impact of EVs in clinical settings, including ischemia-related diseases18, 19. The relevance of EVs in physiological and pathological angiogenesis and their mechanisms of action have been extensively reviewed by Kholia et al.20. EVs that have been physiologically released from several cell types can be detected in biological fluids, including blood21–24. The EVs present in blood could therefore be an important resource for regenerative medicine. Indeed, plasma EVs have been reported to provide a cardioprotective effect against ischemia-reperfusion injury25. Experiments into the depletion of EVs in fetal bovine serum have displayed an impaired ability to induce muscle cell proliferation and differentiation26, suggesting that serum EVs (sEVs) possess defined biological properties. The aim of the present study was to mechanistically investigate the angiogenic potential of EVs that had been purified from the sera of healthy donors (sEVs). In particular, the possible therapeutic impact of sEVs in inducing angiogenesis and improving the reperfusion of acute hind limb ischemia has been investigated. Moreover, an mRNA-angiogenesis microarray has been performed on endothelial cells (ECs) recovered from capillary-like structures formed in response to sEVs in order to dissect their mechanism(s) of action.

Results

Characterization and functional activity of sEVs.  All EV samples were analyzed for their surface antigens via Guava FACS analysis. As expected, it was demonstrated that sEVs are heterogeneous due to their differing cells of origin (platelets, endothelial cells, monocytes/leukocytes) (Supplementary Table S1). The sEV expression of cell adhesion markers was also detected (Supplementary Table S1). Nanosight analyses did not bring to light any significant differences in sEV number and size among the samples. The number of sEVs recovered from a bag of 110 ml was around 6.16 × 108 particles/ml (Supplementary Fig. S1), while sEV diameter was approximately 210 nm with a modal particle size of 183 nm (Supplementary Fig. S1). Starting from 2 × 1011 of total particles, the recovery of sEV RNA and protein was similar in all samples analyzed (n = 18), and ranged from 300 to 500 ng of RNA and 1 to 1.2 µg of protein (Supplementary Fig. S1). To avoid possible contamination of non-sEV-associated mRNAs and proteins, floating density gradient was used to separate sEVs27, 28. The expression of the CD63 exosomal marker was also detected, using Western Blot (WB) analysis, in the 30% iodixanol floating fraction (see Methods) (Supplementary Fig. S2). In order to try and predict the pro-angiogenic activity of sEVs, an in vitro assay was performed using VEGF as the internal control. Effective sEVs (e-sEVs) were considered to be those demonstrating almost 50% of VEGF proangiogenic activity when analyzed for their ability to induce ECs proliferation and tube-like structure formation. As shown in Table 1, 14 out of 18 sEV samples displayed such a feature.

Scientific REPOrTS | 7: 8180 | DOI:10.1038/s41598-017-08250-0

2

www.nature.com/scientificreports/ sEVs induce in vitro and in vivo angiogenesis.  A comparison of in vitro and in vivo experiments was performed in order to validate the predicted sEV pro-angiogenic activity. The in vitro tridimensional angiogenic assay was used to analyze four distinct samples for both e-sEVs and ineffective s-EVs (i-sEVs). As shown in Fig. 1A, e-sEVs were able to induce tube-like structure formation in vitro, unlike i-sEVs. In order to verify these results in vivo, selected sEVs were used in SCID mice. ECs treated with effective and ineffective sEVs (5 × 104 sEV/cell) and mixed with Matrigel were injected subcutaneously into SCID mice for this purpose. Matrigel plugs were removed and analyzed after one week. As shown in Fig. 1B, only e-sEVs displayed pro- angiogenic activity in vivo. These data indicate that our in vitro assays were able to predict sEVs pro-angiogenetic capability. sEVs improve reperfusion recovery and prevent muscle damage after ischemia.  e-sEVs were also evaluated in a murine model of acute ischemic hind limbs that mimics CLI in humans. A total amount of 2 × 1010 e-sEVs was administrated after surgery (see Methods). Different routes of i-sEV and e-sEV administration were assayed in order to evaluate their therapeutic potential (data not shown). The best response, was obtained as follows: intravenous injection immediately after surgery (T0) and intramuscular at T1 and T2. This approach was used throughout the study. Blood perfusion in the ischemic hind limb, as evaluated by Laser Doppler Perfusion imaging immediately after surgery, demonstrated that the ratio between the ischemic and non-ischemic hind limbs decreased in all groups (0.13 ± 0.06 control group; 0.12 ± 0.04 in e-sEV group; and 0.11 ± 0.02 in i-sEV group) (Fig. 2). With the limitation of this approach, which does not allow quantification of deep vascular districts29, 30, we found that e-sEVs, but not i-sEVs and vehicle-treated animals, displayed a significant increased hind limb perfusion ratio at day 7 after surgery (e-sEVs 0.89 ± 0.26 vs control 0.6 ± 0.18 and vs i-sEVs 0.46 ± 0.13, p