EXTRACELLULAR VESICLES IN PATIENT WITH ACUTE CORONARY SYNDROME.
Atherosclerosis associated diseases is the one of the main cause of death in developed countries. Now it is well known that the formation of the atherosclerotic plaque is an inflammatory process in which are involved both cellular and extracellular components. T-cells are one of the main cellular conductor of this process . The study of the pathology of atherosclerotic plaque are complicated by lack of adequate model of monitoring T-cells ex vivo in dynamic. We developed method to cultivating plaques in vitro.
Another crucial component are extracellular – cytokines and extracellular vesicles (EVs)  . Many cell types release EVs into the blood stream, and these EVs may carry proteins characteristic of the cells that released them. Bulk analysis has shown that blood EVs carry tetraspanin CD63, which is expressed by cells of many types and is involved in the EV formation process . Also, it has been reported that two molecules are common to blood EVs: CD41a, a platelet glycoprotein IIb/IIIa that is characteristic of platelets, and CD31, a cell adhesion molecule expressed by endothelia and less abundantly by other cell types.
By incorporating cellular proteins into their membranes, EVs may reflect not only the types of the cells from which they were released but also the physiological state of these cells . Accordingly, EV composition may change in different pathologies, in particular in acute forms of coronary artery disease. The composition of EVs in the blood of patients with acute coronary syndromes (ACS) differs significantly from that found in healthy controls. For study EVs in blood of patient with ACS we developed flow cytometry – based method of phenotyping and enumeration of these particles.
MATERIAL AND METHODS
Peripheral blood was collected by intravenous withdrawal in vacuum tubes with sodium citrate as anticoagulant. Platelet poor plasma (PPP) was prepared within less than 1 h after blood collection by centrifugation at 3,000 g for 15 min. Then, plasma was aliquoted by 600 μl and frozen at -80⁰C.
Coupling of monoclonal antibodies to magnetic nanoparticles
Monoclonal antibodies (Abs) against CD31, CD41a, and CD63 were dissolved in coupling buffer at a concentration of 2 mg/ml. For coupling, 1 mg of 15-nm iron oxide magnetic nanoparticles with carboxylic acid (MNPs) was incubated in 400 µl of activation buffer. Afterwards, 400 µl of coupling buffer was added to activated MNPs followed by the addition of 1 mg of antibody and the mix was incubated for 2 h at room temperature. The reaction was stopped by addition of 10 µl of quenching solution and two washings on strong magnet. In order to visualize nanoparticles, prior to capture of the EVs we mixed the Abs–MNPs with fluorescent anti-mouse IgG1 Fab fragment. We washed away the unbound Fab fragments using a 100-kDa centrifugal device for two 5-min centrifugations at 1,100 g. Labeled Abs–MNPs were diluted in their initial volume with PBS.
Capture and detection of EVs with nanoparticles
PPP (100 µl, freshly thawed) was incubated with 60 µl of labeled Abs–MNPs for 1h at +4⁰ C followed by addition of 2.5% antibody-based blocking reagent. The suspension of EVs conjugated to Abs–MNPs was labeled with fluorescent antibodies or isotype controls for 20 min at room temperature with continuous mixing. For CD31-coupled MNPs we used the following specific monoclonal antibodies: anti-CD41a-APC and anti-CD63-PE. For CD41a-coupled MNPs we used anti-CD31-AlexaFluor® 647 and anti-CD63-PE. For CD63-coupled MNPs we used anti-CD31-PE and anti-CD41a-APC. Afterwards, suspensions were transferred into magnetic columns mounted on an magnet separator. Unbound antibodies were washed away by gravity-mediated flowing of the input sample followed by the flowing of 500 µl of washing buffer for two times (PBS with 2 mM EDTA and 0.5% normal mouse serum). Then, magnetic columns were unmounted and EVs conjugated to Abs–MNPs were eluted with 2 x 400 µl of PBS and fixed with 200 µl of 4% formaldehyde solution. Directly prior to flow cytometry analysis, 50 μl of well-mixed counting beads were added. The procedure is summarized in Fig. 1.
Fig 1. Summary of the protocol for capture and isolation of EVs. MNPs (15 nm) coupled to antibodies against one of EVs’ antigens were incubated with EVs. MNPs were labeled by Fab_AF488 against the capture antibodies. Two other fluorescent antibodies were added to the captured EVs MNP–EV complexes were separated on magnetic columns and analyzed in a flow cytometer.
Flow cytometry analysis
Suspensions of EVs conjugated to Abs–MNPs were analyzed with a FACS Aria II SORP flow cytometer equipped with 355-, 407-, 488-, and 633-nm lasers. Fluorescence activated by the 488-nm laser set at a power of 180.3 mW and measured between 505 and 550 nm (Blue-525-channel) was set as the threshold. The flow cytometer sheath fluid was filtered through a series of in-line filters with nominal filtration rating of 100 nm and 40 nm. First, we acquired filtered PBS and set up the value of the threshold. We set fluorescence parameters in the highest voltage that maintained the positive peak of labeled Compensation Beads in the range of linearity of the trigger channel. We calculated compensation in FlowJo Software using single-stained CompBeads. EV events were counted in a single-event gate according to height and weight parameters of MNP fluorescence.
Cultivating plaque explants
Plaque explants were transported in sterile RPMI 1640 Advanced media with antibiotic/antimicotic. Parts of tissue without calcification or atheromatosis were cut perpendicularly on circular blocks with 2 mm thickness.
Blocks were cultured on collagen sponge in 35 mm petri dishes with 3 ml complete medium based on RPMI 1640 Advanced with added 1%-10mM non-essential amino-acids, 0.2%-100mM sodium pyruvate, Glutamine, antibiotic/antimicotic and 15% fetal bovine serum.
Collagen sponges were washed in complete medium prior using. Plaque were cultured in CO2 incubator on 37°С, 5% CO2. Each 3 days we replace medium.
Flow cytometry of cells from plaque
After weighing we cut tissues on blocks 2mm*2mm*2mm put them in enzyme solution (1,25 mg/ml collagenase from Clostridium histolyticum and 0,2 mg/ml DNAse I) and incubate on 37°С on shaker 1 hour 15 minutes. After, cell suspension was filtered through 40 mkm filter and washed in PBS.
Cells were labeled 15 minutes at room temperature with mixture of antibodies CD45-PE-Cy7, CD3-PerCP-Cy5.5, CD19-PE, CD4-APC-EFluor 780, CD8-eFluor 450. For exclusion of dead cells we used amino reactive dye Alexa Fluor 350.
EVs in blood
We found that in blood of patients as well as in blood of healthy volunteers the most abundant fraction of vesicles was CD41a-positive. We showed that the total amounts of EVs captured by CD41a and positive for one or two of the detection antibodies were significantly higher than the amounts of CD31- and CD63-captured EVs (3,315 [2,411; 6,125] EVs/μl vs. 2,213 [859; 3,661] EVs/μl, p=0.035; vs. 1,908 [707; 3,308] EVs/μl, p=0.003, respectively).
With all the capture strategies used, the total amounts of EVs positive for one or two of the detection antibodies in ACS patients were significantly higher than in healthy volunteers. The amount of EVs captured by CD31–MNPs and positive for one or two of the detection antibodies in ACS patients was 3,359 [2,328; 5,472] EVs/l in comparison with 1,272 [714; 2,157] EVs/l in healthy volunteers (p=0.001). The total amount of EVs captured by CD63 in ACS patients in comparison with healthy volunteers was 3,541 [1,318; 5,173] EVs/μl vs. 806 [488; 2,112] EVs/μl (p=0.007). There were 4,752 [3,238; 7,173] EVs/μl in plasma of patients with ACS captured by CD41a–MNPs, whereas in plasma of healthy volunteers this number was significantly lower, 2,623 [1,927; 4,188] EVs/μl (p=0.015) (Fig. 2). For normally distributed data, we also compared the differences in means and found that in patients there was approximately a 2.4-fold higher amount of CD63-captured EVs (3,207 ± 1,827 vs. 1,321 ± 1,052; p=0.002) and a 1.7-fold higher amount of CD41a-captured EVs (5,296 ± 2,590 vs. 3,069 ± 1,555; p=0.018) than in healthy volunteers.
Fig. 2. Total amounts of EVs in blood of patients with ACS in comparison with healthy volunteers. EVs from patients with ACS (brown symbols) and from healthy volunteers (green symbols) were captured with MNPs coupled to one of three Abs against CD31, CD41a, or CD63 and stained with the other two (fluorescent) Abs. Stained EV–MNP complexes were isolated on magnetic columns and subjected to flow analysis. EVs of different phenotypes have been enumerated. Data presented as dot plot with median and IQR.
We investigated whether the increase in EV concentrations in ACS patient plasma was restricted to a particular fraction of the EVs, or whether the numbers of EVs were proportionally increased in all antigenically different fractions of EVs that we tested. To answer this question, we enumerated EVs in three different subsets for all the captured populations: EVs captured by CD31-MNPs stained for CD41a and CD63, EVs captured by CD63-MNPs stained for CD31 and CD41a, and EVs captured by CD41a-MNP stained for CD31 and CD63.
The amounts of CD31-captured EVs of CD41a+CD63+ or CD41a+CD63− phenotypes were significantly higher in ACS patients than in healthy volunteers (940 [456; 1,415] EVs/μl vs. 342 [246; 622] EVs/μl; p=0.009; and 2,133 [1,764; 4,211] EVs/μl vs. 761 [385; 1,807] EVs/μl; p=0.003, respectively). There were virtually no CD41a-negative vesicles among the CD31-captured EVs (Fig. 3).
Fig. 3. CD31-captured EVs in blood of patients with ACS and healthy volunteers. EVs from patients with ACS (brown symbols) and from healthy volunteers (green symbols) were captured with MNPs coupled to Abs against CD31 and stained with fluorescent Abs against CD41a and CD63. Stained EV–MNP complexes were isolated on magnetic columns and subjected to flow analysis. EVs of different phenotypes have been enumerated. Data presented as dot plot with median and IQR.
There were almost no EVs of CD31+CD41a- phenotype within EVs captured by CD63–MNPs and CD41a+ fractions in ACS patients were significantly larger than in healthy volunteers (2,559 [931; 3,885] EVs/μl vs. 659 [463; 1,708] EVs/μl for CD31+; p=0.009; and 758 [337; 1,282] EVs/μl vs. 147 [36; 568] EVs/μl for CD31-; p=0.012, respectively) (Fig. 4).
Fig. 4. CD63-captured EVs in blood of patients with ACS and healthy volunteers. EVs from patients with ACS (brown symbols) and from healthy volunteers (green symbols) were captured with MNPs coupled to Abs against CD63 and stained with fluorescent Abs against CD41a and CD31. Stained EV–MNP complexes were isolated on magnetic columns and subjected to flow analysis. EVs of different phenotypes have been enumerated. Data presented as dot plot with median and IQR.
In all the fractions of EVs captured by CD41a–MNPs (CD31+CD63-, CD31+CD63+, and CD31-CD63+), we observed increased numbers of EVs in PPP of ACS patients compared with controls. However, only in the CD31+CD63- fraction did this increase (almost two-fold) reach statistical significance (4,356 ± 2,391 EVs/μl vs. 2,199 ± 1,112 EVs/μl; p=0.010) (Fig.5).
Fig. 5. CD41a-captured CD31+CD63- EVs in blood of patients with ACS and healthy volunteers. EVs from patients with ACS (brown symbols) and from healthy volunteers (green symbols) were captured with MNPs coupled to Abs against CD41a and incubated with fluorescent Abs against CD31 and CD63. Stained EV–MNP complexes were isolated on magnetic columns and subjected to flow analysis. EVs of different phenotypes have been enumerated. Data presented as dot plot with mean ± SD.
Plaque explant in vitro cultivating
We found in ruptured plaque 54.65±7.34% CD4+ and 36.99±7.44% CD8+ T-cells compare to non-rupture 45.05±12.74% CD4+ and44.17±12.30% CD8+ T-cells. Ruptured plaques contain significantly high (p<0.05) number CD4+CD8- than non-rupture.
We analyzed T-cells in plaque in dynamic on days 0, 4 and 7. From 0 day to 4 there are tendency to decrease number of cells. After the fourth day the amount of cells are stabilized and live cells even could be detected on day 19 (Fig 6.).
Fig.6. Dynamic of T-cells in cultured plaques. Data represented as M±m
In summary, we found that the predominant population of EVs captured through various antigens carry CD41a, indicating their platelet origin. The amounts of EVs were higher in the ACS patients than in controls. The increase in the numbers of EVs in the ACS patients does not occur across the board in all the EVs with different antigenic compositions, but rather is restricted to CD41a-positive vesicles, probably reflecting activated status of platelets associated with the disease.
We establish method for culturing atherosclerotic plaques in vitro
1.Lymphocytes in Atherosclerosis. Hedrick CC. Arterioscler Thromb Vasc Biol. 2015 Feb;35(2):253-7
2.Inflammatory cytokines in atherosclerosis: current therapeutic approaches. Tousoulis D, Oikonomou E, Economou EK, Crea F, Kaski JC. Eur Heart J. 2016 Feb 2.
3.Extracellular vesicles as mediators of vascular inflammation in kidney disease. Helmke A, von Vietinghoff S. World J Nephrol. 2016 Mar 6;5(2):125-38
4.Colombo M, Moita C, van Niel G, Kowal J, Vigneron J, Benaroch P, et al. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. J Cell Sci. 2013;126: 5553–65
5.György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, et al. Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles. Cell Mol Life Sci. 2011;68: 2667–2688