PDGF-BB carried by endothelial cell-derived extracellular vesicles reduces vascular smooth muscle cell apoptosis in diabetes Gabriele Togliatto1, Patrizia Dentelli1, Arturo Rosso1, Giusy Lombardo1, Maddalena Gili1, Sara Gallo1, Chiara Gai1, Anna Solini2, Giovanni Camussi1*, Maria Felice Brizzi1*
Department of Medical Sciences, University of Torino, Italy
Department of Surgical, Medical, Molecular and Critical Area Pathology, University of Pisa, Italy
GT and PD equally contributed *
Address correspondence to:
Maria Felice Brizzi and Giovanni Camussi, Department of Medical Sciences, University of Turin, Corso Dogliotti 14, 10126, Turin [email protected][email protected] Abstract words: 200 Main text words: 3906 Number of tables/figures: 8 Running Title: mbPDGF-BB in CD31EVs and vascular smooth muscle cell apoptosis Key words: PDGF-BB, extracellular vesicles, diabetes, vascular smooth muscle cells.
Diabetes Publish Ahead of Print, published online January 31, 2018
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therapeutic/prognostic purposes. The roles of CD31EVs as mediators of smooth muscle cell
(VSMC) dysfunction in type 2 diabetes (T2D) is investigated herein.
We demonstrated that, unlike non-diabetic, diabetic serum-derived-EVs (D-CD31EVs) boosted
apoptosis resistance of VSMCs cultured in hyperglycaemic condition. Biochemical analysis
revealed that this effect relies on changes in the balance between anti-apoptotic/pro-apoptotic
signals: increase of bcl-2 and decrease of bak/bax. D-CD31EV cargo analysis demonstrated that D-
CD31EVs are enriched in membrane-bound-platelet-derived-growth-factor-BB (mbPDGF-BB).
Thus, we postulated that mbPDGF-BB transfer by D-CD31EVs could account for VSMC resistance
to apoptosis. By depleting CD31EVs of PDGF-BB or blocking the PDGF-BB-receptorβ on
VSMCs, we demonstrated that mbPDGF-BB contributes to D-CD31EV-mediated bak/bax and bcl-
2 levels. Moreover, we found that bak expression is under the control of PDGF-BB-mediated miR-
296-5p expression. In fact, while PDGF-BB-treatment recapitulated D-CD31EV-mediated anti-
apoptotic program and VSMC resistance to apoptosis, PDGF-BB-depleted CD31EVs failed. D-
CD31EVs also increased VSMC migration and recruitment to neovessels, by means of PDGF-BB.
Finally, we found that VSMCs, from human atherosclerotic arteries of T2D individuals, express
low bak/bax and high bcl-2 and miR-296-5p levels.
This study identifies the mbPDGF-BB in D-CD31EVs as a relevant mediator of diabetes-associated
VSMC resistance to apoptosis.
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Cardiovascular complications are a leading cause of morbidity and premature mortality in
diabetes (1,2). Structural alterations to vessel walls result in intima-media thickening which
individuals at high risk to develop acute cardiovascular events (3,4). Moreover, restenosis is still a
major complication in the diabetic setting. A main cause of re-occlusion is intimal hyperplasia
which is due to the migration and/or excessive growth of vascular smooth muscle cells (VSMCs). A
dysregulated balance between apoptosis and the proliferation of VSMCs seems to play a crucial role
in intima-media thickening in diabetic individuals (5,6). Indeed, in vitro studies have suggested that
high glucose (HG) induces the expression of bcl-2 family members and inhibits the apoptotic
protein Inhibitor of Apoptosis Protein 1, (IAP-1) in VSMCs (7). In addition, Ruiz et al. (8) have
demonstrated that VSMCs, recovered from diabetic patients, show a resistance to apoptosis which
was possibly due to bcl-2 over-expression. Although circulating high glucose concentration might
per se induce VSMC dysfunction, additional events can contribute to this process in vivo.
Several studies have focused on extracellular RNA (exRNA) transporters, indicating that
they may be present in biological fluids in the form of vesicles, which have been denoted
microvesicles, exosomes, membrane particles and apoptotic bodies (9,10). Despite the lack of
consensus on vesicle classification, the presence of overlapping characteristics and biological
activity has evoked the use of the inclusive term; “extracellular vesicles” (EVs) (11,12). The
paracrine/endocrine effects of EVs have recently gained significant attention (13,14). Indeed, EV
biological activity has been linked to the transfer of bioactive molecules, including proteins and
microRNA (miRs) (10-14). EVs are widely distributed in human body fluids, while circulating EV
cargo usually reflects the cell of origin in its physiological and/or pathological condition (9-15).
Indeed, the number and cargo of circulating EVs have been suggested as a means to predict the
presence of disease and even the risk of developing disease (16,17).
Increased levels of circulating platelet- and endothelial cell- (EC) derived microparticles
have been proposed as “biomarkers” of cell dysfunction (18,19). However, EVs might also deliver
specific drivers of disease, as they behave as diffusible vectors of biological activity and participate
in exchanging information. This study therefore investigates the role of EC-derived EVs as
mediators of VSMC fate in type 2 diabetes (T2D).
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RESEARCH DESIGN AND METHODS
Reagents and antibodies are reported in Supplemental Table 1.
Patients and Controls. 11 T2D and 6 non-diabetic individuals (controls), who had undergone
carotid endoarteriectomy surgery in our clinic, were included in the study. Clinical characteristics
are reported in Supplemental Table 2. All diabetic individuals were under statin and metformin
treatment. Ethical approval was obtained from Azienda Ospedaliero-Universitaria (AOU), Città
della Salute e della Scienza di Torino, Italy. Informed consent was obtained from all individuals in
accordance with the Declaration of Helsinki. We had no direct contact with the participants.
Isolation of VSMCs from human atherosclerotic plaque specimens. Human atherosclerotic
plaque specimens were recovered from the above reported subjects (T2D: D; non-diabetic: ND) and
processed as previously described (20). Vascular tissue was rinsed 3 times with phosphate-buffered
saline (PBS) and intima was removed in order to furnish the VSMCs. Tunica media were finely cut
into 2-3 mm pieces and subjected to enzymatic digestion using collagenase type I (0.1mg/ml) in a
Dulbecco’s Modified Eagle Medium (DMEM) for 1.5h at 37°C. Digestion media were collected
and filtered through nylon mesh cell strainers (100µm) to remove the undigested explants. The
resulting supernatants were centrifuged at 1200 rpm for 10 min and cells were plated at 2.5×104
cells/cm2 and cultured with Modified Eagle Medium (MEM) supplemented with 20% (v/v) foetal
REFERENCES 1. Emerging Risk Factors Collaboration. Diabetes mellitus, fasting blood glucose concentration, and risk of vascular disease: a collaborative meta-analysis of 102 prospective studies. Lancet 2010;375:2215–2222 2. Fox CS, Golden SH, Anderson C, Bray GA, Burke LE, de Boer IH Bray GA, Burke LE, de Boer IH, Deedwania P, Eckel RH, Ershow AG, Fradkin J, Inzucchi SE, Kosiborod M, Nelson RG, Patel MJ, Pignone M, Quinn L, Schauer PR, Selvin E, Vafiadis DK; American Heart Association Diabetes Committee of the Council on Lifestyle and Cardiometabolic Health, Council on Clinical Cardiology, Council on Cardiovascular and Stroke Nursing, Council on Cardiovascular Surgery and Anesthesia, Council on Quality of Care and Outcomes Research, and the American Diabetes Association. Update on prevention of cardiovascular disease in adults with type 2 diabetes mellitus in light of recent evidence: a scientific statement from the American Heart Association and the American Diabetes Association. Circulation 2015;132:691–718 3. Terry JG, Tang R, Espeland MA, Davis DH, Vieira JL, Mercuri MF, Crouse JR 3rd. Carotid arterial structure in patients with documented coronary artery disease and disease-free control subjects. Circulation 2003;107:1146-1151 4. Schram MT, Henry RM, van Dijk RA, Kostense PJ, Dekker JM, Nijpels G, Heine RJ, Bouter LM, Westerhof N, Stehouwer CD. Increased central artery stiffness in impaired glucose metabolism and type 2 diabetes: the Hoorn Study. Hypertension 2004;43:176-81 5. Gomez D, Owens GK. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc Res 2012;95:156–164 6. Goel SA, Guo LW, Liu B, Kent KC. Mechanisms of post-intervention arterial remodelling. Cardiovasc Res 2012;96:363-371 7. Li H, Télémaque S, Miller RE, Marsh JD. High glucose inhibits apoptosis induced by serum deprivation in vascular smooth muscle cells via upregulation of Bcl-2 and Bcl-xl. Diabetes 2005;54:540-545 8. Ruiz E, Gordillo-Moscoso A, Padilla E, Redondo S, Rodriguez E, Reguillo F, Briones AM, van Breemen C, Okon E, Tejerina T. Human vascular smooth muscle cells from diabetic patients are resistant to induced apoptosis due to high Bcl-2 expression. Diabetes 2006;55:1243-1251 9. Simons M, Raposo G. Exosomes—vesicular carriers for intercellular communication. Curr Opin Cell Biol 2009;21:575–581 10. Mause SF, Weber C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ Res 2010;107:1047–1057 11. Théry C, Ostrowski M, Segura E. Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 2009;9:581-593 12. Gould SJ, Raposo G. As we wait: coping with an imperfect nomenclature for extracellular vesicles. J Extracell Vesicles 2013;2 13. Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int 2010;78:838-848 14. Ratajczak MZ, Ratajczak J. Horizontal transfer of RNA and proteins between cells by extracellular microvesicles: 14 years later. Clin Transl Med 2016;5:7 15. Yuana Y, Sturk A, Nieuwland R. Extracellular vesicles in physiological and pathological conditions. Blood Rev 2013;27:31-39
16. Chironi G, Simon A, Hugel B, Del Pino M, Gariepy J, Freyssinet JM, Tedgui A. Circulating leukocyte-derived microparticles predict subclinical atherosclerosis burden in asymptomatic subjects. Arterioscler Thromb Vasc Biol 2006;26:2775-2780 17. Ueba T, Nomura S, Inami N, Nishikawa T, Kajiwara M, Iwata R, Yamashita K. Plasma level of platelet-derived microparticles is associated with coronary heart disease risk score in healthy men. J Atheroscler Thromb 2010;17:342-349 18. Sinning JM, Losch J, Walenta K, Böhm M, Nickenig G, Werner N. Circulating CD31+/Annexin V+ microparticles correlate with cardiovascular outcomes. Eur Heart J 2011;32:2034-2041 19. Jung KH, Chu K, Lee ST. Bahn JJ, Kim JH, Kim M, Lee SK, Roh JK. Risk of macrovascular complications in type 2 diabetes mellitus: endothelial microparticle profiles. Cerebrovasc Dis 2011;31:485-493 20. Brizzi MF, Formato L, Dentelli P, Rosso A, Pavan M, Garbarino G, Pegoraro M, Camussi G, Pegoraro L. Interleukin-3 stimulates migration and proliferation of vascular smooth muscle cells: a potential role in atherogenesis. Circulation 2001;103:549-554 21. Togliatto G, Dentelli P, Gili M, Gallo S, Deregibus C, Biglieri E, Iavello A, Santini E, Rossi C, Solini A, Camussi G, Brizzi MF. Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications. Int J Obes (Lond) 2016;40:102-111 22. Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G. Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 2007;110:2440-2448 23. Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, Tetta C, Camussi G. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 2012;7:e33115 24. Greening DW, Xu R, Ji H, Tauro BJ, Simpson RJ. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol 2015;1295:179-209 25. Lombardo G, Dentelli P, Togliatto G, Rosso A, Gili M, Gallo S, Deregibus MC, Camussi G, Brizzi MF. Activated stat5 trafficking via endothelial cell-derived extracellular vesicles controls IL-3 pro-angiogenic paracrine action. Sci Rep 2016;6:25689 26. Gallo S, Gili M, Lombardo G, Rossetti A, Rosso A, Dentelli P, Togliatto G, Deregibus MC, Taverna D, Camussi G, Brizzi MF. Stem Cell-Derived, microRNA-Carrying Extracellular Vesicles: A Novel Approach to Interfering with Mesangial Cell Collagen Production in a Hyperglycaemic Setting. PLoS One 2016;11:e0162417 27. Dentelli P, Barale C, Togliatto G, Trombetta A, Olgasi C, Gili M, Riganti C, Toppino M, Brizzi MF. A diabetic milieu promotes OCT4 and NANOG production in human visceralderived adipose stem cells. Diabetologia 2013;56:173-184 28. Zeoli A, Dentelli P, Rosso A, Togliatto G, Trombetta A, Damiano L, di Celle PF, Pegoraro L, Altruda F, Brizzi MF. Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation. Blood 2008;112:350361 29. Shantikumar S, Caporali A, Emanueli C. Role of microRNAs in diabetes and its cardiovascular complications. Cardiovasc Res 2012;93:583-593 19
30. Wei Y, Schober A, Weber C. Pathogenic arterial remodeling: the good and bad of microRNAs. Am J Physiol Heart Circ Physiol. 2013;304:H1050-1059. 31. Karginov FV, Hannon GJ Remodeling of Ago2-mRNA interactions upon cellular stress reflects miRNA complementarity and correlates with altered translation rates. Genes Dev 2013;27:1624-1632 32. GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national agesex specific all-cause and cause-specific mortality for 240 causes of death, 1990-2013: a systematic analysis for the Global Burden of Disease Study 2013. Lancet 2015;385:117-171 33. Kornowski R, Mintz GS, Kent KM, Pichard AD, Satler LF, Bucher TA, Hong MK, Popma JJ, Leon MB. Increased restenosis in diabetes mellitus after coronary interventions is due to exaggerated intimal hyperplasia. A serial intravascular ultrasound study. Circulation 1997;95:1366-1369 34. Brooks MM, Jones RH, Bach RG. Chaitman BR, Kern MJ, Orszulak TA, Follmann D, Sopko G, Blackstone EH, Califf RM. Predictors of mortality and mortality from cardiac causes in the bypass angioplasty revascularization investigation (BARI) randomized trial and registry. For the BARI Investigators. Circulation 2000;101:2682-2689 35. Henry RM, Kostense PJ, Dekker JM, Nijpels G, Heine RJ, Kamp O, Bouter LM, Stehouwer CD. Carotid arterial remodeling: a maladaptive phenomenon in type 2 diabetes but not in impaired glucose metabolism: the Hoorn study. Stroke 2004;35:671-676 36. Watson PA, Nesterova A, Burant CF, Klemm DJ, Reusch JE. Diabetes-related changes in cAMP response element-binding protein content enhance smooth muscle cell proliferation and migration. J Biol Chem 2001;276:46142-46150 37. Yasunari K, Kohno M, Kano H, Yokokawa K, Minami M, Yoshikawa J. Antioxidants improve impaired insulin-mediated glucose uptake and prevent migration and proliferation of cultured rabbit coronary smooth muscle cells induced by high glucose. Circulation 1999;99:1370-1378 38. Hall JL, Matter CM, Wang X, Gibbons GH. Hyperglycemia inhibits vascular smooth muscle cell apoptosis through a protein kinase C-dependent pathway. Circ Res 2000;87:574580 39. Sakuma H, Yamamoto M, Okumura M, Kojima T, Maruyama T, Yasuda K. High glucose inhibits apoptosis in human coronary artery smooth muscle cells by increasing bcl-xL and bfl-1/A1. Am J Physiol Cell Physiol 2002;283:C422-C428 40. Karch J, Molkentin JD. Regulated necrotic cell death: the passive aggressive side of Bax and Bak. Circ Res 2015;116:1800-1809 41. Guay C, Regazzi R. Circulating microRNAs as novel biomarkers for diabetes mellitus. Nat Rev Endocrinol 2013;9:513-521 42. Togliatto G, Dentelli P, Brizzi MF. Skewed Epigenetics: An Alternative Therapeutic Option for Diabetes Complications. J Diabetes Res 2015;2015:373708 43. Gu J, Zhang H, Ji B, Jiang H, Zhao T, Jiang R, Zhang Z, Tan S, Ahmed A, Gu Y. Vesicle miR-195 derived from Endothelial Cells Inhibits Expression of Serotonin Transporter in Vessel Smooth Muscle Cells. Sci Rep. 2017;7:43546 44. Grotendorst CR, Chang T, Seppa HEJ, Kleinman HK, Martin GR. Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells. J Cell Physiol 1982;113:261– 266 20
45. Bilato C, Pauly RR, Melillo G, Monticone R, Gorelick-Feldman D, Gluzband YA, Sollott SJ, Ziman B, Lakatta EG, Crow MT. Intracellular signaling pathways required for rat vascular smooth muscle cell migration. Interactions between basic fibroblast growth factor and platelet-derived growth factor. J Clin Invest 1995;96:1905-1915 46. McNamara CA, Sarembock IJ, Bachhuber BG, Stouffer GA, Ragosta M, Barry W, Gimple LW, Powers ER, Owens GK. Thrombin and vascular smooth muscle cell proliferation: implications for atherosclerosis and restenosis. Semin Thromb Hemost 1996;22:139-144 47. Andrae J, Gallini R, Betsholtz C. Role of platelet-derived growth factors in physiology and medicine. Genes Dev 2008;22:1276-1312 48. Raines EW. PDGF and cardiovascular disease. Cytokine Growth Factor Rev 2004;15:237254 49. Weber A, Köppen HO, Schrör K. Platelet-derived microparticles stimulate coronary artery smooth muscle cell mitogenesis by a PDGF-independent mechanism. Thromb Res 2000;98:461–466 50. Goetzl EJ, Schwartz JB, Mustapic M, Lobach IV, Daneman R, Abner EL, Jicha GA. Altered cargo proteins of human plasma endothelial cell-derived exosomes in atherosclerotic cerebrovascular disease. FASEB J 2017;31:3689-3694 51. Tüfekci KU, Meuwissen RL, Genç S. The role of microRNAs in biological processes. Methods Mol Biol 2014;1107:15-31 52. Barile L, Vassalli G. Exosomes: Therapy delivery tools and biomarkers of diseases. Pharmacol Ther 2017;pii:S0163-7258(17)30034-7 53. Pant S, Hilton H, Burczynski ME. The multifaceted exosome: biogenesis, role in normal and aberrant cellular function, and frontiers for pharmacological and biomarker opportunities. Biochem Pharmacol 2012;83:1484-1494
vs ND-CD31EVs). (D-E) An in vitro angiogenesis assay was performed, as above, using HG-EVs
control siRNA or HG-EVs depleted of PDGF-BB (PDGF-BB siRNA) (scale bars=20µm, 40X
magnification). Data are reported in the histogram as number±SEM of VSMCs per number of
tubular structures (p<0.001, HG-EVs PDGF-BB siRNA vs HG-EVs control siRNA and none, n=5).
(F) VSMC migration assay was performed in HG conditions under the indicated treatment (n=5,
20X magnification) (p=0.009, HG-EVs PDGF-BB siRNA vs HG-EVs control siRNA).
Representative images were acquired on a confocal microscope.
Figure 5. VSMCs from T2D individuals express high levels of bcl-2 and low bak/bax content.
(A) Representative FACS analysis of CD31 and alpha-smooth muscle cell (α-SMA) surface
markers expressed by VSMCs recovered from T2D (D, n=11) and non-diabetic (ND) individuals
(ND, n=6). All data are reported in the Table (mean percentage±SD). Isotype control was included.
(B) bak/bax and bcl-2 content was analyzed on all ND- or D-VSMC samples, normalized to α-SMA
content. The statistical analysis of all samples (D, n=11; ND, n=6) is reported in (C) (p<0.01, D vs
ND for bak, p<0.001, D vs ND for bax, p<0.05, D vs ND for bcl-2).
Figure 6. VSMC miR-296-5p expression is increased in hyperglycaemic condition and boosted
by PDGF-BB (A) The indicated miRs were evaluated by qRT-PCR in VSMCs recovered from
T2D (D) and ND human atherosclerotic plaque specimens. Data normalized to RNU6B are
representative of all samples (D, n=11; ND, n=6) (p=0.02, D vs ND for miR-296-5p). (B) miR‐29624
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5p was evaluated by qRT‐PCR on LG- or HG-treated VSMCs and normalized to RNU6B (p=0.03,
HG- vs LG-treated VSMCs, n=6). (C) Loss-of-function experiments were performed on LG- and
HG-cultured VSMCs for 48h, using antago-miR control or antago-miR-296-5p oligonucleotides.
After 48h cells were lysed and analyzed for bak/bax and bcl-2 content, normalized to α-SMA
(p<0.001, LG-antago-miR control and LG-anti-miR-296-5p vs HG-antago-miR control for bak/bax
and bcl-2; p<0.001, HG-antago-miR control vs HG-antago-miR-296-5p for bak and bcl-2; p<0.05,
HG-antago-miR control vs HG-antago-miR-296-5p for bax, n=3). (D) Apoptosis assay was
performed on VSMCs cultured and treated as in (C). Doxorubicin (1µmol/l) served as positive
control (c+). Data are expressed as percentage±SEM (n=5) of total apoptotic cells (p<0.001, LG-
antago-miR control vs HG- antago-miR control, HG- antago-miR control vs HG-antago-miR-296-
5p; p<0.001, all experimental conditions vs control, c+). (E) miR‐296-5p expression, normalized to
RNU6B, was evaluated by qRT‐PCR in LG and HG-cultured VSMCs both with and without
PDGF-BB (10 ng/ml) (p=0.002, PDGF-BB vs none in HG-treated VSMCs, n=6). (F) miR‐296-5p
expression, normalized to RNU6B, was evaluated by qRT‐PCR in HG-cultured VSMCs, treated as
indicated (p=0.007, HG-EVs control siRNA vs none; p=0.002, HG-EVs PDGF-BB siRNA vs HG-
EVs control siRNA, n=6).
Figure 7. miR-296-5p post-transcriptionally regulates bak expression. (A) Blast analysis of hsa-
miR-296-5p sequence and BAK1 3′UTR full-length shows a base pairing from 1138 to 1158 bp. (B)
pGL3 empty vector and pGL3-3′UTR BAK1 luciferase constructs were transfected into LG- and
HG-cultured VSMCs. Relative luciferase activity is reported (p<0.001 HG vs LG in pGL3–3′UTR
BAK1-transfected cells, n=5). (C) pGL3 and pGL3-3′UTR BAK1 constructs were transfected into
LG-cultured VSMCs previously transfected with pre-miR control or with pre-miR-296-5p. Relative
luciferase activity is reported (p<0.001 pre-miR-296-5p vs pre-miR control in pGL3–3′UTR BAK1
transfected cells, n=5). (D) Bak content was analyzed on cell extracts from VSMCs overexpressing
miR-296-5p (pre-miR-296-5p), not transfected or transfected with pGL3 or pGL3-3′UTR BAK1 25
constructs and normalized to α-SMA (p<0.001 VSMCs/pre-miR-296-5p none and pGL3 vs
VSMCs/pre-miR-296-5p+pGL3-3′UTR BAK1, n=5).
Figure 8. Schematic representation of HG and D-CD31EV mechanism of action. ND-
CD31EVs do not affect VSMC fate due to their low mbPDGF-BB content (left panel). In the
diabetic setting, D-CD31EVs enriched in mbPDGF-BB content affect VSMC fate by promoting
resistance to apoptosis (right panel).
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Figure 1 rev3
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Figure 2 rev3
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Figure 4 rev3
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Figure 5 rev3
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Figure 7 rev3
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Figure 8 rev3
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On line SUPPLEMENTAL DATA. Supplemental Table1. Reagents and Antibodies. Reagents Description: Purchased from: Bovine calf serum (BSA) (A8806), collagenase IA (C9891), FCS (12138C), FBS (F6178), RNase (R6513), SDS (L3771), PIPES (P9291), Triton X-100 (T8787), Nonidet P-40 (74385), NaCl (S3014), NaF (S7920), Na3OV4 (S6508), Na4P2O7 (P8010), MgCl2 (M8266), KCl (P9541), HCl (258148), Na-azide (S2002), Hepes (H3375), Tris (T1503), EDTA (E6758), EGTA Sigma-Aldrich (St Louis, MO, USA) (E4378), ethanol (51976), aprotinin (A6279), pepstatin A (P5318), PMSF (P7626), DMSO (D8418), PKH26 dye (MINI26-1KT), PKH67 dye (MIDI67-1KT), leupeptin (L2884), penicillin-streptomycin (P4333), DMEM 5 mmol/l (D5546), DMEM 25 mmol/l (D5671), M199 (M4530), DAPI (4',6-diamidino-2-phenylindole) Protein molecular weight markers (1610374), Acrylammide (161-0156), polyvinylidene difluoride (PVDF) Bio-Rad (Hercules, CA, USA) membranes (162-0115), Bradford reagent (500-0205), ECL (170-5061) BD Bioscience Pharmingen (Franklin Lakes, Matrigel Basement Membrane Matrix NJ, USA) Growth Factor Reduced (356231) MEM (31095-029), Lipofectin® Reagent (18292-037), TRIZOL (15596018), hsa-miR296-5p (000527), RNU6B (001093), antiInvitrogenTM (Life Technologies Carlsbad, CA, miR-296-5p (AM10609), pre-miR-296-5p USA; Paisley, UK). (PM10609), hsa-miR-24-3p (000402), hsamiR-222 (002276), hsa-miR-221 (000524) Syber Green primers: RNU6B: CGCAAGGATGACACGCAA miR-21-5p: TAGCTTATCAGACTGATGTTGA Quiagen (Valencia, CA, USA) miR-29a: TAGCACCATCTGAAATCGGTTA miR-145;GTCCAGTTTTCCCAGGAATCC Human recombinant IL-3 (200-03) PeproTech EC Ltd (London, UK) Human recombinant PDGF-BB (100-14B) Antibodies Description: Purchased from: anti-CD31-APC human (130-092-652) Miltenyi Biotec, Auburn, CA, USA anti-CD14-PE human (130-110-519) anti-PDGFR β (AF385) Human PDGF-BB Quantikine Elisa R&D Systems, MN, USA (DBB00) anti-β actin (sc-47778) S. Cruz Biotechnology (Heidelberg, Germany) anti-PDGF-BB (ab178409) for WB anti bcl-2 (ab59348) Abcam (Cambridge, UK) anti bak (ab32371) anti bax (ab32503)
anti-bcl2l2 (ab38629) anti CD63 (ab134045) anti α-SMA (ab32575) anti-VEGFA (ab46154) anti-rabbit IgG, HRP linked (4050-05) anti-mouse IgG, HRP linked (1031-05) anti CD42b-FITC human (555492) anti-mouse IgG-PE (550589) anti-mouse and rabbit IgG-FITC (555988 and 554020)
Southern Biotech (Birmingham, Alabama USA)
BD Bioscience Pharmingen (Franklin Lakes, NJ, USA)
Cell cultures. Primary macrovascular endothelial cells (ECs) and VSMCs were purchased from Lonza (Basel, Switzerland) and cultured as described by the manufacturer’s instructions. VSMCs and ECs were used at II-III cell-culture passage. To collect the EVs, ECs were starved in DMEM under either low (LG, 5mmol/l) or high glucose (HG, 25 mmol/l) and 24h deprived of bovine calf serum (BCS). Cell viability was evaluated using trypan blue at the end of each experiment (94±5% viable cells/experiment). In selected experiments, siRNA technology was performed in HG-cultured ECs by transfecting either the siRNA negative control or the Platelet-Derived Growth Factor (PDGF-BB) siRNA (Applied Biosystems) for 48h (1). EV isolation was then obtained from HGcultured ECs depleted of PDGF-BB. In selected experiments, VSMCs were cultured for 48h in LG or HG DMEM and then treated without BCS for 24h in the presence of ND-CD31EVs, DCD31EVs or EC-derived-EVs (5×103 for all types of EVs/target cell), as indicated. Alternatively, HG-cultured VSMCs were stimulated for 24h with PDGF-BB (10 ng/ml). In selected experiments, HG-cultured VSMCs were pre-incubated for 30 min at 37°C with a blocking PDGF-Receptorβ (PDGFRβ) antibody (5µg/ml). All experiments were performed in accordance with European Guidelines and policies and approved by the Ethical Committee of the University of Turin. Isolation and characterization of EVs and CD31EVs from sera of T2D and non-diabetic individuals. Human serum from all above T2D and non-diabetic individuals was obtained before surgery and after informed consent. EVs from each participant were obtained by centrifuging serum as previously described (2). The supernatant was subsequently submitted to differential ultracentrifugation at 10k and 100k g for 2h at 4°C. EVs pellets were then re-suspended in DMEM: final volume of 100 µl plus 1% of DMSO and stored at -80°C. FACS analysis of D-EVs and ND-
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EVs was performed as indicated in (3), using anti-CD31-allophycocyanin (APC), anti-CD14phycoerythrin (PE) and anti-CD42b-FITC antibodies. FITC, PE or APC mouse non-immune isotypic IgG (BD Bioscience Pharmingen) were used as controls. FACS analysis was performed using a Guava easyCyteTM Flow Cytometer (Millipore, Germany). Fluorochrome conjugated antibodies were added to a suspension of EVs (2.5x106 particles/100 µl) for 15 min at 4°C. The volume was increased to 500 µl using a FACS flow and the expression of surface markers was evaluated. Surface marker expression is reported in the representative histograms as the percentage of expression±SD. The CD31 microbead kit (Miltenyi Biotec, Auburn, CA, USA) was used to isolate CD31EVs from the sera of T2D (D-CD31EVs) and non-diabetic individuals (NDCD31EVs) (4). Briefly, 0,5 ml of freshly-thawed plasma was incubated with 100 µl of CD31 microbeads for 4h at 4°C. EVs captured on CD31 Ab-coated magnetic beads were recovered from the magnetic column (MS column) via elution with rinsing solution, as described in the manufacturer’s instructions. EV-bound beads were submitted to differential ultracentrifugation at 10k and 100k g (Beckman Coulter Optima L-90K ultracentrifuge; Beckman Coulter, Fullerton, CA) for 3h at 4°C. CD31EVs were either used fresh or were stored at -80°C and then processed for transmission electron microscopy (TEM), biological effects, western blot and q-RT-PCR analysis. CD63 content in CD31EVs was analyzed by western blot. Transmission electron microscopy (TEM). TEM was performed on CD31EVs that had been isolated by ultracentrifugation and re-suspended in PBS, placed on 200 mesh nickel form//var carbon coated grids (Electron Microscopy Science, Hatfield, PA) and left to adhere for 20 min. Grids were then incubated with 2.5% glutaraldehyde, containing 2% sucrose, and EVs were negatively stained with NanoVan (Nanoprobes, Yaphank, NK, USA), after being washed in distilled water, and observed under a Jeol JEM 1010 electron microsco pe (Jeol, Tokyo, Japan). Isolation of EC-derived EVs. ECs were cultured in LG or HG DMEM without BCS for 24h, in order to collect the EVs from supernatants. After being centrifuged at 3k g for 30 minutes, in order to remove debris, cell-free supernatants were submitted to differential ultracentrifugation at 10k and
100k g (Beckman Coulter Optima L-90K ultracentrifuge) for 3h at 4°C. EVs were either used fresh or were stored at -80°C after re-suspension in DMEM supplied with 1% Dimethyl sulfoxide (DMSO) (v/v). Frozen EVs were washed and pelleted under 100k g ultracentrifugation to remove DMSO before cellular experiments. No difference in biological activity was observed between fresh and stored EVs. The protein content of EVs was quantified using the Bradford method (Bio-Rad, Hercules, CA, USA). Any possible contamination was tested for using a Limulus amebocyte assay (concentration <0.1 ng/ml) (Charles River Laboratories, Inc., Wilmington, MA, USA). EV number and size distribution analysis was performed using a NanoSight LM10 (NanoSight Ltd, Minton Park UK). The particles in the samples were illuminated using a laser light source and the scattered light was captured by camera and analyzed using Nanoparticle Tracking Analysis (NTA). NTA automatically tracked and sized particles according to Brownian motion and the diffusion coefficient (Dt). Results were displayed as number per ml and as a frequency size distribution graph, outputted to a spreadsheet. EC-derived EVs were then processed for biological, western blot and q-RT-PCR analysis (1,2). EV internalization. The internalization of EVs was evaluated using confocal microscopy (LSM5PASCAL; Zeiss, Oberkochen, Germany) as previously described (5). A pool of approximately 2.5x108 EV particles was labeled with red fluorescent PKH26 dye (2 µl/ml) for 30 min at 37°C and then EVs were washed and were submitted to differential ultracentrifugation at 10k and 100k g for 1h at 4°C. EV pellets were suspended in M199 and added to HG-cultured VSMCs (2x104) pretreated or not with a blocking PDGFRβ antibody in order to detect their internalization (5). Z-stack confocal microscopy VSMC images were also obtained. Western blot analysis. Cells and EVs were lysed and protein concentrations were obtained, as previously described (5). 50 µg proteins, for cells, and 10 µg, for EVs, were subjected to Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred into nitrocellulose membranes and processed as previously described (5). Densitometric analysis was used to calculate
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the differences in the fold induction of protein levels which were normalized to α-SMA, β-actin or CD63 content. Values are reported as relative amounts. RNA isolation and quantitative real-time PCR (qRT-PCR) for miRs. Total RNA was isolated from the VSMCs of atherosclerotic plaque specimens and from human VSMCs, that had been treated as indicated or left untreated, using the TRIzol reagent (Invitrogen). Total RNA was also isolated from the different EVs using the mirVana RNA isolation kit (Ambion), according to manufacturer’s instructions. RNA was quantified spectrophotometrically (Nanodrop ND-1000, Wilmington, DE, USA) because intact 18S and 28S rRNAs were difficult to detect in the EVs. RNA from cells and EVs was then reverse-transcribed using a TaqMan or a Syber Green microRNA RT kit, specific for miR-24-3p, miR-221, miR-222 and miR-296-5p, or a Syber Green microRNA RT Kit specific for miR-21-5p, miR-29a and miR-145, as indicated. Thus, RNA was subjected to qRT-PCR using a TaqMan microRNA assay kit and the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA, USA). miR expression was normalized to the small nuclear RNA, RNU6B. Loss- and gain-of-function experiments were performed in VMSCs that had been transfected for 48h with the antago-miR negative control, the anti-miR-2965p antagomir, pre-miR negative control or pre-miR-296-5p oligonucleotides (Applied Biosystem), according to manufacturer’s instructions (6). Luciferase miRNA target reporter assay. The luciferase reporter assay was performed using a construct generated by subcloning the PCR products amplified from the full-length 3’UTR of human BAK1 DNA into the Xba restriction site of the luciferase reporter vector pGL3 (Promega, Madison, WI, USA). The PCR products were obtained using the following primers for BAK1: sense,5’TTTCTAGATGACTCCCAAGGGTGCCCTT3’; antisense,5’AATCTAGATGAGGGGATTGCACAGTT3’. The insert identities were verified by sequencing. The pGL3, pGL3-3’UTR BAK1 luciferase reporter vectors were transiently cotransfected in VSMCs, treated as indicated, at 10:1 molar ratio with the pRL vector, coding for the Renilla luciferase, used as internal control of the luciferase assay (6). Luciferase activities, using the
pGL3 reporter vectors, above described, were also evaluated in VSMCs transfected with pre-miR296-5p. Cell proliferation and apoptosis assay. Proliferative activity was assayed via direct cell count by 3 individual operators in triplicate, as previously described (number±SEM of cells per field, 20X magnification) (7). For the apoptosis assay, VSMCs, which had been cultured under different experimental conditions, were subjected to Muse Annexin V and the cell dead assay (Merck, Darmstadt, Germany) in accordance with manufacturer’s instructions. Tubule-like structure formation assay. In order to analyze the EC/VSMC interaction, 24-wellplates were coated with growth factor-reduced Matrigel matrix (BD Biosciences) to analyze tubulelike structure formation (8). Briefly, HG-cultured ECs and VSMCs were pre-treated, for 24h, with either D-CD31EVs, ND-CD31EVs or HG-EC-EVs that had either been depleted, or not, of PDGFBB. 4.5x104 red labeled ECs (PKH26 vital dye) were placed in HG medium on top of the polymerized matrix. 2x104 green labeled VSMCs (PKH67 vital dye) were then added to ECs. Cocultures were thus incubated at 37°C and non-adherent cells were removed via washing three times with phosphate-buffered saline. After 6h, the number of VSMCs/tubule-like structures formed and the number of green-labeled VSMCs were counted by three different operators (each experiment was performed in triplicate), counting 10 fields at 40X magnification. Images were acquired on a Zeiss LSM 5 Pascal confocal laser-scanning microscope (Carl Zeiss). Scratch assay on VSMCs. Scratch assays were performed on HG-cultured VSMCs, treated as indicated, in order to evaluate cell migration activity. VSMCs were seeded to a final density of 100,000 cells per well and maintained at 37 °C and 5% CO2 for 24h in order to allow cell adhesion and the formation of a confluent monolayer to occur. These confluent monolayers were then scored using a sterile pipette tip to leave a scratch of approximately 0.4–0.5 mm in width. The culture medium was then immediately removed (along with any dislodged cells). The removed medium was replaced with a fresh, serum-free culture medium for 24h either in the presence or absence of the indicated EVs. Image acquisition was performed on a phase-contrast microscope at times 0 and
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24h later by 3 individual operators in triplicate (20X magnification). Images were analyzed using the Leica QWin v3.0 program. Data are reported in the histogram as the difference in scratch length in µm (∆) observed at time 0 and at time 24h to indicate the distance covered by the cells. ELISA Assays. PDGF-BB concentration in D-CD31EVs were measured using a commercially available competitive enzyme immunoassay (ELISA) kit (R&D Systems, MN, USA), according to manufacturer's instructions. To evaluate mbPDGF-BB, intact or lysates D-CD31EVs (2.5x108 particles) were compared. The same samples were pre-treated with trypsin (0.25%) for 1h and blocked with BSA 1% (negative control). After trypsin treatment supernatants were submitted to differential ultracentrifugation at 10k and 100k g (Beckman Coulter Optima L-90K ultracentrifuge) for 1h at 4°C and analyzed.
REFERENCES 1. Togliatto G, Dentelli P, Gili M, Gallo S, Deregibus C, Biglieri E, Iavello A, Santini E, Rossi C, Solini A, Camussi G, Brizzi MF. Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications. Int J Obes (Lond) 2016;40:102-111 2. Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G. Endothelial progenitor cell derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood 2007;110:2440-2448 3. Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, Tetta C, Camussi G. Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 2012;7:e33115 4. Greening DW, Xu R, Ji H, Tauro BJ, Simpson RJ. A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. Methods Mol Biol 2015;1295:179-209 5. Lombardo G, Dentelli P, Togliatto G, Rosso A, Gili M, Gallo S, Deregibus MC, Camussi G, Brizzi MF. Activated stat5 trafficking via endothelial cell-derived extracellular vesicles controls IL-3 pro-angiogenic paracrine action. Sci Rep 2016;6:25689 6. Gallo S, Gili M, Lombardo G, Rossetti A, Rosso A, Dentelli P, Togliatto G, Deregibus MC, Taverna D, Camussi G, Brizzi MF. Stem Cell-Derived, microRNA-Carrying Extracellular Vesicles: A Novel Approach to Interfering with Mesangial Cell Collagen Production in a Hyperglycaemic Setting. PLoS One 2016;11:e0162417
7. Dentelli P, Barale C, Togliatto G, Trombetta A, Olgasi C, Gili M, Riganti C, Toppino M, Brizzi MF. A diabetic milieu promotes OCT4 and NANOG production in human visceralderived adipose stem cells. Diabetologia 2013;56:173-184 8. Zeoli A, Dentelli P, Rosso A, Togliatto G, Trombetta A, Damiano L, di Celle PF, Pegoraro L, Altruda F, Brizzi MF. Interleukin-3 promotes expansion of hemopoietic-derived CD45+ angiogenic cells and their arterial commitment via STAT5 activation. Blood 2008;112:350361
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Supplemental Table2. Clinical characteristics of type 2 diabetic (T2D) and non-diabetic individuals enrolled in the study.
Total triglycerides 106.2±37.77 (mmol/l)
Total cholesterol 132±19.21 (mmol/l)
Supplemental Figure S1
Supplemental Figure S1. (A) Cell extracts from low glucose (LG)- and high glucose (HG)-treated VSMCs were analyzed for bak/bax and bcl-2 content, normalized to α-SMA (p<0.05, HG- vs LGtreated VSMCs for bak, p<0.001, HG- vs LG-treated VSMCs for bax and bcl-2, n=5). (B) Cell extracts from low glucose (LG)- and high glucose (HG)-treated VSMCs were analyzed for bcl2l2, normalized to α-SMA (n=3). (C) The Mouse Annexin V dead cell kit (apoptosis assay) was used. Doxorubicin (1µmol/l) was used as a positive control (c+). Data are expressed as percentage±SEM (n=6) of total apoptotic cells (p<0.001, LG- and HG-treated vs positive control (c+); p=0.03, HGvs LG-treated VSMCs). (D) Cell proliferation assay was performed in VSMCs treated for 48h, as indicated, and reported as number of cells per field (mean±SEM, 20X magnification). Interleukin-3 (IL-3), 10 ng/ml, was used as a positive control (c+) (n=6) (p<0.001, t0, LG- and HG-treated vs positive control (c+)).
Supplemental Figure S2
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Supplemental Figure S2. (A) Representative images (EVs recovered from ECs cultured in LG or HG conditions: LG- and HG-EVs) of NanoSight analyses on the 100k fraction of Curve 1, relationship between particle distribution (left y axis) and particle size (x axis); curve 2, correlation between cumulative percentage distribution of particles (percentile in right y axis) and particle size (x axis). (B) PDGF-BB content was analyzed in LG- and HG-EVs and normalized to CD63 (p=0.001, HG-EVs vs LG-EVs, n=6). (C) VEGF content was analyzed in LG- and HG-EVs and normalized to CD63 (n=4). (D) miR‐296-5p expression, normalized to RNU6B, was evaluated by qRT‐PCR on EVs recovered from LG- or HG-treated ECs (LG- and HG-EVs) (p=0.002, HG-EVs
vs LG-EVs, n=6). (E) miR‐296-5p was evaluated by qRT‐PCR on D-CD31EVs and ND-CD31EVs. Data normalized to RNU6B are representative of all samples (D, n=11; ND, n=6) (p=0.02, DCD31EVs vs ND-CD31EVs).
Supplemental Figure S3
Supplemental Figure S3. Loss-of-function experiments were performed on LG- and HG-cultured VSMCs for 48h, using antago-miR control or antago-miR-296-5p. miR-296-5p expression was evaluated by qRT-PCR and normalized to RNU6B (p<0.001, antago-miR-296-5p vs antago-miR control in LG and HG, n=5).
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PDGF-BB carried by endothelial cell-derived extracellular vesicles ...
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PDGF-BB carried by endothelial cell-derived extracellular vesicles reduces vascular smooth muscle cell apoptosis in diabetes G...
May 31, 2018 - Diseases, University of Notre Dame, Notre Dame, IN, United States of America, 2 Department of Electrical. Engineering and Computer Science, ...
cellular vesicles (EVs) such as exosomes and microvesicles (MVs) (2). Accordingly, patients with vascular diseases associated with systemic endothe-.
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Nov 21, 2016 - SHRSP arteries, EDH-mediated hyperpolarization and relaxation were significantly impaired when compared with WKY. GSK1016790A, a selective TRPV4 activator, evoked robust hyperpolarization and relaxation in WKY arteries. In contrast, in
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Abstract. Microglia are the primary immune effector cells resident within the CNS, whose activation into migratory, phagocytic cells is associated with increased expression of cell adhesion molecules of the integrin family. To determine which specifi