METHOD FOR CHARACTERIZATION OF CELL SPECIFIC MICROVESICLES

Abstract

The present invention refers to a method for characterizing and/or measuring and/or sorting the amount of cardiac-derived microvesicles, having the step of detecting CD172a marker on the microvesicles, in an isolated biological sample obtained from the subject.

Claims

1. An ex-vivo or in vitro method for characterizing and/or measuring the amount of subsets of circulating tissue-derived microvesicles (MV) comprising the step of detecting the CD172a marker on said microvesicles, in an isolated biological sample obtained from the subject, wherein if the microvesicle is positive for CD172a the microvesicle is characterized as a cardiac derived microvesicle.

2. The ex-vivo or in vitro method according to claim 1 further comprising the step of further detecting at least one marker selected from the group consisting of: CD235a, CD61, CD144, CD14, CD45, CD73, CD3 and combinations thereof.

3. The method according to claim 1 wherein the markers to be detected are CD172a, CD235a, CD61, CD144, CD14, CD45 and CD73.

4. The method according to claim 1 wherein: if the microvesicle is positive for CD235a the microvesicle is characterized as an erythroid derived MV; if the microvesicle is positive for CD61 the microvesicle is characterized as a platelet derived MV; if the microvesicle is positive for CD144 the microvesicle is characterized as an endothelium-derived MV; if the microvesicle is positive for CD14 the microvesicle is characterized as a monocyte-derived MV; if the microvesicle is positive for CD45 the microvesicle is characterized as a leukocyte derived MV; and if the microvesicle is positive for CD73 the microvesicle is characterized as a stromal/adipocyte derived MV.

5. An ex-vivo or in vitro method for diagnosing and/or assessing the risk of developing and/or prognosing and/or for monitoring the progression and/or for monitoring the efficacy of a therapeutic treatment and/or for the screening of a therapeutic treatment of cardiovascular diseases (CVD), in a subject comprising the steps of: a) characterizing and/or measuring the amount of subsets of circulating tissue-derived microvesicles according to the method of claim 1; and b) comparing with respect to a proper control and/or reference.

6. The method according to claim 5, wherein an amount of cardiac-derived MV, in the isolated biological sample obtained from the subject, higher than the control amount indicates that the subject is either affected by or is at increased risk for developing cardiovascular diseases (CVD).

7. The method according to claim 5, wherein an amount of cardiac-derived MV, in the isolated biological sample obtained from the subject, lower than the control amount indicates that the subject is going toward an amelioration of the CVD.

8. The method according to claim 7, wherein the subject undergone a valve replacement.

9. The method according to claim 5 wherein the cardiovascular disease (CVD) is selected from the group consisting of: heart failure, aortic stenosis (AS), valvular disease, cardiomyopathy, acute coronary artery disease (CAD), atherosclerosis, myocardial ischemia, infarction, arrhythmias, hypertrophic cardiomyopathy (HCM) and drug-dependent cardiotoxicity connected to different pathologies.

10. The method according to claim 5 wherein the measured or characterized subset of circulating tissue-derived microvesicles is the cardiac-derived MV subset.

11. The method according to claim 10 wherein the cardiac-derived MV subset is characterized by the presence of the marker CD172a.

12. The method according to claim 11 wherein the cardiac derived microvesicles are negative for the following markers: CD235a, CD61, CD144, CD14, CD45 and CD73.

13. The method according to claim 5 wherein the isolated biological sample is one of plasma, blood, serum, tissue obtained by surgical resection, tissue obtained by one of biopsy, cells culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow.

14. The method according to claim 1 wherein the marker is detected by means of a specific ligand.

15. The method according to claim 14 wherein the ligand is an antibody, or functional fragment thereof.

16. The method according to claim 14, wherein the marker is detected by magnetic beads coated with antibody capture and/or customized dried antibody cocktails and/or columns with sized filter cartridges and/or combined with specific antibody filter (SAF).

17. The method according to claim 1 wherein the subsets of circulating tissue-derived microvesicles are characterized and/or their amount measured by means of multicolor flow cytometry technology (FACS), immunogold electron microscopy, immunofluorescence, ELISA, immunoprecipitation, reverse colorimetric immunoassay (RCIA), radioimmune assay (MA) Electrochemiluminescence, surface plasmon resonance (SPR)-based approach, nanoliter microfluidics (immunoassays) or spectometry.

18. (canceled)

19. (canceled)

20. A kit comprising detecting means for carrying out the method of claim 1.

21. The method according to claim 8, wherein the valve replacement is a transcatheter aortic valve implantation (TAVI).

22. The method according to claim 9, wherein the heart failure is primary or secondary.

23. The method according to claim 9, wherein the pathology is cancer, HiV-HAART therapies.

Description

[0164] The invention will be now illustrated by means of non-limiting examples referring to the following figures.

[0165] FIG. 1. Stepwise gating strategy by FACS of circulating tissue-derived MV subsets.

[0166] (a) FACS analysis of plasma derived MV subpopulations assessed by immunostaining. (b) Difference in vesicle size between SYTOX+ apoptotic bodies (R3), cell membrane fragments or apoptotic bodies Phallotoxin+/SYTOX (R2) and total microvesicles Phallotoxin/SYTOX (R5+R7+R8+R10+R11+R13+R14) was confirmed comparing their morphological distribution with Megamix beads at the same FSC/SSC values. Representative images out of three independent experiments. (c) Analysis of Annexin V expression on circulating tissue-derived MV subsets. Images are representative of four analyzed patients.

[0167] FIG. 2. Absolute count of plasma derived microvesicle subpopulations.

[0168] (a-c) Flow cytometric absolute count of SYTOX+ apoptotic bodies (R3), cell membrane fragments or apoptotic bodies Phallotoxin+/SYTOX (R2) and total microvesicles Phallotoxin/SYTOX (R5+R7+R8+R10+R11+R13+R14). (b-c) Flow cyto metric absolute count of microvesicle subpopulations R5, R7, R8, R10, R11, R13, and R14. In all panels the MV absolute count was performed using Trucount beads. The analysis were performed on healthy donor (n=4) and TAVI patients before and upon two months of Follow Up (n=3-5). (a-b) Unpaired and (c) paired Wilcoxon test; (*P<0.05; **P<0.01). Error bars represent SEM.

[0169] FIG. 3. Cardiac-derived microvesicle in AS patients with preselected cut-off.

[0170] (a-b) Flow cytometric absolute count of microvesicle subpopulations R13 in patients with pre-selected cut-off (CD172a-derived MV absolute count/mL < and >4.7). Wilcoxon Matched Pairs analysis was performed on n=10 (4.7) and n=11 (<4.7). (*P<0.05; **P<0.001). Error bars represent SEM. (c) R13 ROC area against gold standard (NT-proBNP).

[0171] FIG. 4. CD172a+ MVs drive cardiac specific molecules.

[0172] (a) Representative intracellular flow cytometry analysis of cardiac troponin T expression on CD172a+ microvesicles. Platelet derived microvesicles (CD61+) were used as internal negative control. Representative images out of three independent observations. (b) Real time PCR analysis of miR-1, miR-133a and miR-21 in sorted CD172a+ microvesicles. Sorted monocyte derived microvesicles (CD14+) were used as internal negative controls for the cardiac specific miR-1 and miR-133a (nd=not detectable). Data shown are from three independent observations. Error bars represent SEM.

[0173] FIG. 5. CD172a+ MVs in healthy donors.

[0174] Flow cytometric analysis of the cardiac derived MVs (CD172a+) obtained from a control group stratified for gender (female (F), male (M)) and age (years (Y)<65; Y65). The MV absolute count was performed using Trucount beads. Error bars represent SEM.

[0175] FIG. 6. CD172a+ MVs in Aortic stenosis patients.

[0176] Flow cytometric analysis of the cardiac derived MVs obtained from healthy donors (HD) (n=52) and Aortic Stenosis (AS) patients (n=109). The MV absolute count was performed using Trucount beads. Mann Whitney test; (***P<0.001). Error bars represent SEM.

[0177] FIG. 7. CD172a+ MVs in Aortic stenosis patients before and upon 1 year of TAVI surgery.

[0178] (a) Flow cytometric absolute count of cardiac derived microvesicles (CD172a+) (R13) (left panel) and other total MVs (R5+R7+R8+R10+R11+R13) (right panel) in Aortic Stenosis (AS) patients before and upon 1 year of TAVI surgery (n=109). In all panels the MV absolute count was performed using Trucount beads. (b) Kaplan-Meier Survival Curves performed between Groups identified by cut off. Mann-Whitney U-test (*P<0.05; Error bars represent SEM) and Long rank mantel cox test respectively (**P<0.01).

[0179] FIG. 8. CD172a+ MVs in Aortic stenosis Italian patients before and upon TAVI surgery.

[0180] Flow cytometric absolute count of cardiac derived microvesicles (CD172a+) (R13) (top panel) and other total MVs (R5+R7+R8+R10+R11+R13) (bottom panel) in Aortic Stenosis (AS) patients before and upon 48 h and 2 months of TAVI surgery (n=10). In all panels the MV absolute count was performed using Trucount beads. Mann-Whitney U-test; (*P<0.05). Error bars represent SEM.

[0181] FIG. 9. CD172a+ MVs in cardiac pathological conditions

[0182] Flow cytometric absolute count of cardiac derived microvesicles (CD172a+) in Healthy Donors (HD) (n=52) Hypertrophic Cardiomyopathy (HCM) (n=15) and Coronary Artery Disease (CAD) (n=28) patients. MV absolute count was performed using Trucount beads. In order to verify the selective changes in Cardiac MV absolute counts in each pathological condition analyzed, data were normalized using the following formula: Cardiac MVs (R13)/Total other MVs (R5+R7+R8+R10+R11+R14)*1000. The multiplier was arbitrarily selected in order to better illustrate the data in decimal range>0. (per 1000). Mann-Whitney U-test; (*P<0.05; **P<0.01). Error bars represent SEM.

EXAMPLES

Example 1

[0183] Materials and Methods

[0184] Plasma Isolation and Storage

[0185] According to published recommendations, plasma sampling and storage techniques were standardized for overall patients and healthy subjects.

[0186] Informed consent was obtained from each patient and healthy subjects.

[0187] A 5 ml sample of peripheral blood was collected in EDTA-containing Vacutainer tubes. The vials were processed within 2 h of collection by centrifugation at 1200g at room temperature in a bench top centrifuge for 20 minutes to eliminate all blood cells. To further reduce leukocyte and red cells contamination, the top third of the plasma was aspirated and placed in fresh tubes and frozen at 80 C.

[0188] MV Isolation from Plasma.

[0189] Human plasma was diluted with filtered PBS/ (no calcium and magnesium) and centrifuged at 500 g30 minutes at 4 C. The obtained Platelet Free Plasma (PFP) was centrifuged at 12000 g45 minutes at 4 C. Finally, the supernatant was removed leaving 25 l of a MV-enriched suspension which was further diluted with 75 l of filtered PBS/. [Caby M P, et al. Int Immunol. 2005 July; 17(7):879-87]

[0190] Circulating MV Characterization and Count by FACS

[0191] To limit background noise from dust and crystals, a 0.22 m filtered sheath fluid was indifferently used preserving sterility, for sample acquisition.

[0192] A morphological gate of microvesicles was performed using Megamix Plus FSC beads (0.1 m to 1 m beads Biocytex), according to literature [Mobarrez F, et al., Thromb Res. 2010 March; 125:e110-6].

[0193] Events included in a range from 0.1 m to 5 m were clearly discriminated from background noise.

[0194] Apoptotic bodies and possible cell membrane fragments derived from freezing/thawing procedure were stained using SYTOX (Invitrogen-Molecular Probes) and Phallotoxin (Invitrogen-Molecular Probes) at room temperature (RT) for 15 and 30 minutes, respectively [Mobarrez F, et al., Thromb Res. 2010 March; 125:e110-6].

[0195] In detail, the SYTOX molecule is a high affinity nucleic acid dye while the Phallotoxin binds F-actin. Both dyes easily penetrates only compromised plasma membranes.

[0196] According to the present stepwise gating strategy, only events SYTOX/Phallotoxin double negatives were analyzed for the detection of tissue-derived MV using the appropriate saturating concentrations of highly specific surface antibodies (30 minutes at RT).

[0197] Tissue-Derived MV Surface Antibodies Selection.

[0198] The antibodies were selected according to literature, as following: [0199] R5 Subset: CD235a (GA-R2) (BD Biosciences); [Van Beers E J, et al. Haematologica.2009 November; 94(11):1513-9] [0200] R7 Subset: CD61 (VI-PL2) (BD Biosciences); [Crompot E, et al. PLoS One. 2015 May 15; 10(5):e0127209] [0201] R8 Subset: CD144 (REA199) (Miltenyi Biotech); [Koga H, et al. J Am Coll Cardiol. 2005 May 17; 45(10):1622-30] [0202] R10+R11 Subset: CD45 (HI30) (BD Biosciences) combined with CD14 (M5E2) (BD Biosciences); [Thtinen S, et al. Cancer Immunol Res. 2015 May 14] [Griffin J D, et al J. Clin. Invest. 1981; 68: 932-41]. [0203] R14 Subset: CD73 (AD2) (BD Biosciences); [Mller G, et al. Obesity (Silver Spring). 2011 August; 19(8):1531-44] [0204] R13 Subset: CD172a (15-414) (eBioscience), not yet published its involvement in circulating cardiac-derived MVs. [Dubois N C, et al. Nat Biotechnol. 2011 Oct. 23; 29(11):1011-8]

[0205] To avoid potential co-expression of CD172a on monocyte- and leukocyte-MVs derived, as previously reported in the literature, the present method enables to discriminate monocyte- and leukocyte-MVs derived (R10 and R11) in the first steps of the matrix.

[0206] All gate regions were restrictively defined using both negative controls: Fluorescence Minus One (FMO) and isotype controls.

[0207] Internal MV Quality check: Phosphatidylserine (PS), a marker on the extracellular leaflet of MVs, was verified in all characterized MV-subsets with saturating concentration of Annexin V (BD Biosciences) for 15 minutes at RT.

[0208] Circulating MV absolute count was performed using Trucount beads (BD Biosciences), following manufacturer's instructions.

[0209] Results

[0210] The present inventors' stepwise gating strategy, reported in FIG. 1A, is a potent and strategic tool able to accurately discriminate the uninvestigated cardiac (R13)-derived MV, minimizing the false positive events and using an anti-CD172a antibody not known as marker for circulating cardiac-derived MV.

[0211] The first assessed gate allows the identification of ABs (R3) and/or membrane fragments (R2). The sequential gating strategy is able to exclude the predominant MV erythroid (R5), platelet (R7), endothelium (R8) and leukocyte (R10 and R11) and to specifically focus on cardiac-(R13) and stromal/adipocyte (R14) -derived MV.

[0212] As showed in FIG. 1b, these three main subpopulation were size compared with Megamix Plus FSC beads and, as expected, R2 and R3 had a dimension>0.5 m while the other MVs (R5;R7;R8;R10;R11;R13;R14) ranged between 0.1 and 0.5 m.

[0213] Moreover the matrix 77 of the antibodies, reported in Table 1, is representative of the applied stepwise gating strategy. Noteworthy, the high specificity of the antibodies with a cross reaction <10% and accuracy of system (100% for cardiac-derived MV) were successfully reached.

[0214] Internal MV Quality check: The Annexin V, a surface marker for the microvesicles [Heijnen H F, et al Blood. 1999 Dec. 1; 94(11):3791-9.], was evaluated in overall characterized MV subsets, as showed in FIG. 1c.

[0215] Preliminary Data on CVD Patients

[0216] 1) Pilot Study

[0217] Plasma samples were collected from patients with aortic stenosis (AS) at the time of inclusion (time=0) from Federico II University, Naples. AS patients were then undergone percutaneous aortic valve replacement (TAVI). Clinical follow-up and plasma sampling was performed at a 2-months follow-up (time=2M). Five TAVI patients and four healthy subjects were recruited.

[0218] Our stepwise gating strategy was applied and the absolute count was summarized in FIG. 2. Among the MV characterized from plasma, AB (R3), erythroid-(R5), endothelium-(R8) and cardiac-(R13) derived MV subsets were found at significantly higher levels in pre-TAVI AS patients as compared with healthy donors (FIGS. 2a and 2b). Strikingly, levels of circulating cardiac- and endothelium-derived MV subsets were significantly reduced after TAVI (FIGS. 2a and 2b).

[0219] Noteworthy, the lower levels of AB (R3) and endothelium-(R8) derived MVs were confirmed applying a Wilcoxon Matched Pairs test (FIG. 2c) while the cardiac (R13)-derived MV subset was slightly under the significant threshold, probably due to small sample size.

[0220] 2) Improvement of Preliminary Data in Independent AS Patients

[0221] The same findings were obtained increasing the sample size with an independent group of AS patients enrolled from the University Hospital of Kiel, Germany. The present stepwise gating strategy was applied on 21 pre TAVI AS patients followed at 7 days and 365 days post-surgery. Strikingly, levels of circulating cardiac- and endothelium-derived MV subsets were significantly reduced after 365 days of follow up. Significant Wilcoxon Matched Pairs p-values were obtained. Monocitic- and stromal-derived showed the same significant trends after 1 years post-surgery.

[0222] Additionally, the significant interlinking between cardiac, endothelium-, monocitic- and stromal-derived MV was observed by Spearman' rho correlation analysis.

[0223] In detail, the interlinked MV-MV showed a rho0.80 a P<0.0000, as reported in the correlation matrix of Table II.

[0224] The identified inter-linking between MV-MV may characterize also other CVDs.

[0225] To optimize and identify a potential grey zone of cardiac biomarkers (CD172a) identifying for (R13)-derived MV, a threshold analysis was performed. A quintile-based method and Time-based strategy.

[0226] A similar cut-off was obtained, as following reported:

[0227] Quintile-based method: cut-off4.7 (absolute count/mL), AUC: 0.51 IC95%: 0.34-0.65, Std Error 0.07, Sensitivity (Se): 56% and Specificity (Sp): 48%;

[0228] Inventors obtained Se and Sp<0.70 but for biomarkers monitoring disease progression (as well as drug responsiveness) the patient serves as his own control (baseline values versus follow-up values), and the best Se or Sp threshold (>0.7, etc) becomes less important.

[0229] Inventors selected a R13 cut-off to accurately identify the potential Grey Zone (MV absolute count<4.7/mL) and the best performer cut-off of our cardiac-biomarker (MV absolute count4.7/mL). The threshold of the cardiac-biomarker able to discriminate patients with AS was optimized in the phase of the updated data using a larger cohort of AS patients and age-matched control group.

[0230] According to previous data about the interlinking of MV-MV, the best performer cut-off of cardiac biomarkers (4.7) enables to identify the best performer cut-off and the grey zone of the other correlated MV (endothelium-, monocitic- and stromal-derived MVs).

[0231] Wilcoxon Matched Pairs analysis was performed into two obtained groups (CD172a cut-off<(n=11) and 4.7 absolute count/mL (n=10), respectively). (FIG. 3a-FIG. 3b) A strikingly reduction of 72% of CD172a was observed post 365 days.

[0232] Finally test equality of ROC area against gold standard (NT-proBNP) was performed in patients with pre-designed CD172a cut-off (4.7 absolute count/mL). (FIG. 3c) Cardiac-biomarkers showed the higher AUC=0.8727 (Std. Err. 0.08) than gold standard AUC=0.7455 (Std. Err. 0.12).

[0233] Tables

TABLE-US-00001 TABLE 1 The matrix 7 7 of Stepwise gating strategy to characterize circulating tissue-derived MVs from Human Plasma. % CD235a.sup.+ CD61.sup.+ CD144.sup.+ CD14.sup.+ CD45.sup.+ CD172a.sup.+ CD73.sup.+ R5 100 5.5 0.8 12.1 4.7 2.3 0.8 2 0.2 2.7 0.7 8.6 3.8 R7 0 100 0 1.3 0.3 0.3 0.1 0.6 0.3 1.5 0.4 R8 0 0 100 2.5 0.7 2.7 0.6 3.3 1.5 4 0.8 R10 0 0 0 100 3 0.6 4.2 1.7 1.9 0.8 R11 0 0 0 0 100 8.7 3.2 7 2 R13 0 0 0 0 0 100 0 R14 0 0 0 0 0 0 100

[0234] Cross reaction or/and co-expression of the antibodies used in the panel were put in a matrix. Data shown are the mean of six samples (Standard Error).

[0235] R5: erythroid-derived; R7: platelet-derived; R8: endothelium-derived; R10: monocyte-derived; R11: leukocyte-derived; R13: cardiac-derived; R14: stromal/adipocyte-derived circulating MVs.

TABLE-US-00002 TABLE II Inter linking MV-MV CD172a CD144 CD73 CD14 CD172a 1 CD144 Rho = 0.81 1 P < 0.0000 CD73 Rho = 0.83 Rho = 0.92 1 P < 0.0000 P < 0.0000 CD14 Rho = 0.84 Rho = 0.94 Rho = 0.95 1 P < 0.0000 P < 0.0000 P < 0.0000

[0236] Data shown are the P and the Spearman's CorrelationCoefficient (r)s of tissue derived-MVs in AS patients.

Example 2

[0237] MV Isolation and Storage.

[0238] According to published recommendations, plasma samples and storage techniques were standardized. A 5 ml sample of peripheral blood was collected in EDTA-containing Vacutainer tubes. The vials were processed within 2 h of collection by centrifugation at 1,200 g20 min. at room temperature (RT) to eliminate all blood cells. To further reduce leukocyte and red cell contamination, the top third of the plasma was aspirated and placed in a fresh tube. According to the literature, isolated plasma was subsequently diluted with filtered PBS and centrifuged at 500 g30 min. at 4 C. The obtained platelet-free plasma was centrifuged at 12,000 g45 min. at 4 C. Finally, the supernatant was removed, leaving 25 l of an MV-enriched suspension, which was diluted with 75 l of filtered PBS (Caby M P, et al. Int Immunol. 2005 July; 17(7):879-87).

[0239] FACS Analysis.

[0240] To limit background noise from dust and crystals, a 0.22 m filtered sheath fluid was used for sample acquisition. A morphological gate of microvesicles was performed according to Megamix-Plus FSC beads size (range: 0.1-0.9 m) (Biocytex), according to literature (Mobarrez F, et al., Thromb Res. 2010 March; 125:e110-6). Events included in a range from 0.1 m to 5 m were clearly discriminated from background noise. Apoptotic bodies and cell membrane fragments derived from freezing/thawing were stained using SYTOX (Invitrogen-Molecular Probes) and Phalloidin (Invitrogen-Molecular Probes) at RT for 15 and 30 minutes, respectively (Mobarrez F, et al., Thromb Res. 2010 March; 125:e110-6). Only SYTOX/Phalloidin events were analyzed for the detection of tissue-derived MVs using the appropriate saturating concentrations of the following conjugated monoclonal antibodies: CD235a (BD Biosciences) (Van Beers E J, et al. Haematologica.2009 November; 94(11):1513-9); CD61 (BD Biosciences) (Crompot E, et al. PLoS One. 2015 May 15; 10(5):e0127209); CD144 (Miltenyi Biotech) (Koga H, et al. J Am Coll Cardiol. 2005 May 17; 45(10):1622-30); CD45 (BD Biosciences) (Thtinen S, et al. Cancer Immunol Res. 2015 May 14); CD14 (BD Horizon) (Griffin J D, et al J. Clin. Invest. 1981; 68: 932-41); CD3 (BD Biosciences) (Jones et al. 1993; Kothlow et al. 2005); CD73 (BD Biosciences) (Ode A, Eur Cell Mater. 2011 Jul. 6; 22:26-42) (F. Barry, R. Biochemical and Biophysical Research Communications, vol. 289, no. 2, pp. 519-524, 2001); and CD172a (eBioscience) (Dubois N C, et al. Nat Biotechnol. 2011 Oct. 23; 29(11):1011-8). MV quality check: Phosphatidylserine (PS) expression was verified in all characterized MV subsets using saturating concentration of annexin V (BD Biosciences).

[0241] Monoclonal anti-cardiac troponin T (cTnT) (Innova Biosciences) was used to detect intracellular cTnT in CD172a+ MVs as well as in CD61+ MVs, as an internal negative control, with the Intrasure KiT (BD Biosciences), according to the manufacturer's instructions.

[0242] All gated regions were restrictively defined using Fluorescence Minus One (FMO) and/or isotype controls as negative controls. Circulating MV absolute count was obtained with BD Trucount tube (IVD-CE, BD Biosciences), following the manufacturer's protocol. An LSRFortessa analyzer (BD Bioscience) was used for sample acquisition. Data acquisition and analysis were performed with FACSDivav.6.2 (BD Biosciences) and Flow-jo v.9.7 (Tree Star Inc.), respectively. In order to minimize the time-dependent changes in MV count (Lrincz M, J Extracell Vesicles. 2014 Dec. 22; 3:25465), the stability of MV quantification was evaluated in five healthy subjects at two time points: baseline (time=0) with fresh isolated MVs, and after one month (time=1M) storage at 80 C. No differences were observed between the two time-points analysed, suggesting that isolated MVs can be stored for short periods (up to 1M).

[0243] MV Sorting.

[0244] Circulating MVs were sorted using a FACSAria III cell sorter (Biosciences) equipped with 4 lasers and able to discriminate up to 18 fluorescences. MV sorting was validated using the MegaMix bead (BioCytex). The cell sorter instrument was optimized to sort 0.3 m, 0.5 m and 0.9 m beads with a purity higher than 95%.

[0245] Cardiac-Specific MicroRNA (miRNA) Analysis in Sorted Tissue-Derived MVs

[0246] Total RNA from sorted cardiac-derived MVs was extracted with TRIzol reagent (Invitrogen, Carlsbad, Calif., USA) following the standard protocol. Reverse transcription reactions were performed using the EXIQON miRNA Reverse Transcription Mercury Universal cDNA synthesis kit (Exiqon A/S, Vedbaek, Denmark). Specific primer sequences of cardiac-specific miRNAs (miR-1 [miRBase ID: MIMAT0000416] and miR-133a [miRBase ID: MIMAT0000427]) and miR-21a [miRBase ID: MIMAT0000076], which is not exclusively heart-derived, were obtained (www.exiqon.com/microrna-real-time-per-primer-sets) according to literature (Car et al. Nat Med. 2007 May; 13(5):613-8; Catalucci et al. Circ Cardiovasc Genet. 2009 August; 2(4):402-8). The Freedom Evo 150 liquid handling system (Tecan Group, Mannedorf, Switzerland) was used for aliquoting reaction mixtures and samples in all protocol steps. qRT-PCR was performed with a 7900HT Fast Real-Time PCR (Applied Biosystems). miRNA content was evaluated in cardiac-derived MVs as well as in monocyte- and leukocyte-derived MVs, as negative controls, in experimental settings. The miRNA Ct cut off value was considered 39.

[0247] Patients Enrollment.

[0248] All subject has been enrolled, conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the Ethical Review Board. Informed consent was obtained from each patient. Enrollment was conducted at Humanitas Clinical and Research Institute, University Hospital of Kiel, Germany, Federico II University of Naples, Clinica Mediterranea (Naples, Italy) and AVIS Comunale Milano, (Milan, Italy).

[0249] Aortic Stenosis (AS) Cohort.

[0250] Patients with Severe symptomatic aortic stenosis (aortic valve area, AVA, <1 cm2; body surface indexed AVA, iAVA, <0.6 cm2/m2) (Svensson L G, Adams D H, Bonow R O, et al. Aortic valve and ascending aorta guidelines for management and quality measures. Ann Thorac Surg 2013; 95:S1-66) were recruited at the University Hospital of Kiel, Germany (n=109) and Federico II University of Naples (n=10).

[0251] AS patients were then undergone percutaneous aortic valve replacement (TAVI). All subjects underwent physical examination, electrocardiogram (ECG), 24-hour ECG Holter monitoring, TTE and Doppler studies, and CMR.

[0252] Plasma samples were collected from patients with aortic stenosis (AS) at the time of inclusion (time=0). Clinical follow-up and plasma sampling was performed at 48 h, 2 months and 365 days post-surgery.

[0253] Negative outcomes during FU period were also recorded.

[0254] Hypertrophic Cardiomyopathy (HCM) Cohort.

[0255] 15 unrelated patients diagnosed with HCM were recruited at the Division of Cardiology, Federico II University of Naples, Naples, Italy. The diagnosis of HCM was based on echocardiographic demonstration of a hypertrophied, non-dilated LV (wall thickness>15 mm) in the absence of any other cardiac or systemic disorder producing a comparable grade of hypertrophy (Gersh B J, Maron B J, Bonow R O, et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2011; 124:27861-94). Classification parameters defining HCM status were those established by the American Heart Association guidelines (Gersh B J, Maron B J, Bonow R O, et al. 2011 ACCF/AHA guideline for the diagnosis and treatment of hypertrophic cardiomyopathy: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines. Circulation 2011; 124:2761-96). None of the patients was in heart failure.

[0256] CAD Cohort.

[0257] 28 patients scheduled between Jan. 7, 2008 and Jan. 31, 2010 to undergo elective DES [drug-eluting stents] implantation at the Clinica Mediterranea (Naples, Italy) were assessed for their suitability for the study. Exclusion criteria were: either non-ST-segment or ST-segment elevation myocardial infarction; cardiogenic shock; allergy/intolerance to aspirin and/or clopidogrel; ongoing serious bleeding or bleeding diathesis; platelet count 75.000/mm3; planned or undelayable noncardiac surgery; previous percutaneous coronary intervention or coronary artery bypass grafting; severe liver disease (e.g., cirrhosis or portal hypertension); and life expectancy<1 year due to other medical conditions. Diabetes mellitus (DM) was diagnosed according to current guidelines (American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2004; 27 Suppl 1:S5-10). Chronic kidney disease (CKD) was defined as an estimated glomerular filtration rate<60 ml/min/1.73 m2 (National Kidney Foundation. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. AmJ Kidney Dis 2002; 39 Suppl 1:S1-266).

[0258] Control Group.

[0259] 52 healthy subjects were recruited by AVIS (Milan), a multicenter Italian blood donor organization, and Humanitas Research and Clinical Center (Rozzano). Additionally, individuals without cardiovascular disease were obtained from the South Italian Centenarian Study. (Anselmi C V, et al. Rejuvenation Res. 2009 April; 12(2):95-104). None of the selected healthy subjects had a family history of cardiovascular disease.

[0260] Statistical Methods.

[0261] Normality assumption was checked. To evaluate differences between MV-count subsets, Wilcoxon matched pairs test or Mann-Whitney U-test were applied as appropriate. The optimum cutoff value was obtained according to Youden Index (J) and quintile-based method. Clinical Endpoint defined as death was estimated in our cardiac-derived MV by Kaplan-Meier product limit estimator. Comparison of Survival Curves was performed using log-rank Mantel-Cox test. Statistical significance was defined as 2-sided p<0.05 for all tests. The statistical analyses were performed using STATA 11/SE (College Station, Tex.) and GraphPad PRISM 5.

[0262] Results

[0263] Inventors identified CD172a as surface marker for the identification of cardiac derived MVs in plasma of Aortic Stenosis (AS) patients. In order to further address the cardiac origin of these MVs inventors analysed their internal content by investigating the expression of both cardiac specific Troponin T and micro RNAs. The flow cytometric analysis performed on permeabilized circulating MVs revealed a selective expression of cardiac troponin T in the CD172a+ subpopulation (FIG. 4a). Moreover, this result was supported by the real time PCR analysis for Cardiac specific miRNAs performed on sorted CD172a+ MVs. As showed in FIG. 4b, the CD172a+ MVs expressed the cardiac specific miR1 and miR133a and the non-cardiac specific miR21 while the CD14+ MVs (internal negative control) expressed only the non-cardiac specific miR21. Altogether these results confirmed the cardiac origin of CD172a+ MVs.

[0264] Inventors showed a significant decrease of cardiac MVs (CD172a+) in Aortic Stenosis (AS) patients upon TAVI surgery suggesting the release of these MVs from cardiac cells under stress conditions. In order to compare the absolute count of CD172a+ MVs in AS patients with an enlarged cohort of healthy donors (HD), inventors firstly evaluated the cardiac derived MVs in healthy donors (n=52) stratified for gender and age. In detail, the control group included n.32 individuals aged <65 years (n.8 females and n.24 males; range years: 31-64) and n.2065 years (n.14 females and n.6 males; range years: 66-98). Noteworthy, the absolute count of circulating CD172a+ MVs showed any significant age- and gender-related link, as reported in FIG. 5. Further inventors compared the absolute count of CD172a+ MVs observed in HDs with a cohort of AS patients (n=109) (FIG. 6).

[0265] Data obtained revealed significant higher level in AS patients' as compared to HDs (***P<0.001) confirming the results obtained by the present inventors.

[0266] In order to strengthen the data of the cardiac MVs (CD172a+) release in Aortic Stenosis (AS) patients upon 1 year of TAVI surgery (shown in the Example 1 section), the original cohort was increased for a total of n.109 AS patients. The obtained data showed the selective reduction of the cardiac MVs (*P<0.05) upon TAVI surgery together with no significant fluctuation of the total MVs analysed (FIG. 7a).

[0267] According to defined cut-off (CD172a+ MV absolute count=1,79/ml), two AS groups at time of the inclusion was identified: Group A with CD172a+ MV absolute count>=1,79/ml and B with CD172a+ MV absolute count<1,79/ml. Interestingly, the negative outcome rate (i.e. death) was higher in Group B than in Group A (**P<0.01) (FIG. 7b). These findings suggest a potential prognostic relevance of circulating CD172a+ MVs for TAVI treatment (i.e. responder/not responder).

[0268] To verify the robustness of the present strategy and the potential role of cardiac-derived MVs as biomarker of myocardial stress, an independentAS cohort was included in the study.

[0269] Circulating tissue-derived MVs of n. 10 patients with AS undergone percutaneous aortic valve replacement from Federico II University, Naples were evaluated at time of inclusion (pre-TAVI) up to 2-months follow-up, as summarized in FIG. 8. The significant reduction of circulating cardiac-derived MV subset was confirmed 2 months after surgery.

[0270] Finally, in the effort to better define the pathogenesis of the CD172a+ MV release, two different pathological phenotypes characterized by cardiac suffering: Hypertrophic CardioMyopathy (HCM) (n=15) and Coronary Artery Disease (CAD) (n=28) patients were enrolled. Interestingly, the CD172a+ MVs appeared selectively increased as compared to HDs (*P<0.05 and **P<0.01 respectively) (FIG. 9).