Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy
Abstract
:1. Introduction
2. Results
2.1. Cytokine Levels in Blood Plasma and Conditioned Medium from Atherosclerotic Plaques
2.2. The Choice and Verification of the Normalization Control Cytokines
2.3. Comparison of the Normalized Cytokine Levels in Blood Plasma and Conditioned Medium from Atherosclerotic Plaques
2.4. Target Chemokine Gene Expression in Atherosclerotic Plaques
2.5. Target Chemokine Gene Expression in Different Cell Subsets of Atherosclerotic Plaques and Peripheral Blood
3. Discussion
4. Materials and Methods
4.1. Patients
4.2. Blood Plasma and Conditioned Culture Medium
4.3. xMAP Cytokine Measurement
4.4. xMAP Data Analysis
4.5. Cell Sorting
4.6. RNA Extraction
4.7. Real-Time qPCR
4.8. Data Analysis
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A
Patient | Sex | Age, Years | Artery | Max Stenosis, % | History of Ischemic Stroke (year) | History of Transient Ischemic Attack (year) | Signs of Fibrous Cap Rupture (CT or Macroscopic Examination during Surgery) | Specimen Used for |
---|---|---|---|---|---|---|---|---|
1 | Female | 69 | Right CCA bifurcation, ICA | 90 | No | No | - | Luminex analysis |
2 | Male | 65 | Left CCA bifurcation, ICA | 65 | No | No | - | Luminex analysis |
3 | Male | 57 | Right CCA bifurcation, ICA | 90 | No | No | No (macroscopic examination) | Luminex analysis |
4 | Male | 51 | Right ICA | 90 | Yes (2020) | No | - | Luminex analysis |
5 | Female | 76 | Right CCA bifurcation, ICA | 90 | No | No | - | Luminex analysis |
6 | Female | 78 | Left CCA bifurcation, ICA, ECA | 90 | No | No | - | Luminex analysis |
7 | Male | 69 | Right ICA | 70 | No | Yes (2017) | - | Luminex analysis |
8 | Male | 69 | Right ICA | 70 | No | No | - | Luminex analysis |
9 | Male | 55 | Left ICA | 80 | No | No | Yes (CT) | Luminex analysis |
10 | Male | 55 | Right ICA | 90 | Yes (2017, 2018) | No | - | Luminex analysis |
11 | Female | 60 | Left ICA | 90 | No | No | Yes (macroscopic examination) | Luminex analysis |
12 | Male | 62 | Left ICA | 70 | No | No | Yes (macroscopic examination) | Luminex analysis |
13 | Male | 71 | Left ICA | 75 | No | No | No (macroscopic examination) | Luminex analysis |
14 | Female | 58 | Right CCA bifurcation, ICA | 70 | No | No | No (macroscopic examination) | Luminex analysis |
15 | Female | 74 | Right CCA bifurcation, ICA | 90 | No | No | No (macroscopic examination) | Luminex analysis |
16 | Female | 64 | Left ICA | 70 | No | No | - | Luminex analysis |
17 | Male | 61 | Left ICA | 75 | No | No | No (macroscopic examination) | Luminex analysis |
18 | Male | 57 | Left CCA bifurcation, ICA | 90 | No | No | Yes (macroscopic examination) | Luminex analysis |
19 | Male | 62 | Left CCA bifurcation, ICA | 70 | No | No | No (macroscopic examination) | Luminex analysis |
20 | Female | 55 | Right CCA bifurcation, ICA | 70 | No | No | No (macroscopic examination) | Luminex analysis |
21 | Male | 69 | Left CCA bifurcation, ICA | 75 | Yes (2021) | No | No (macroscopic examination) | Luminex analysis |
22 | Female | 67 | Right ICA | 85 | No | No | Yes (macroscopic examination) | Luminex analysis |
23 | Male | 65 | Left ICA | 85 | No | No | - | Luminex analysis |
24 | Male | 72 | Left ICA | 90 | Yes (2023) | No | Yes (macroscopic examination) | qPCR, bulk, sorted cells |
25 | Male | 67 | Right ICA | 95 | Yes (2015) | No | No (CT) | qPCR, bulk, sorted cells |
26 | Male | 63 | Left ICA | 70 | Yes (2023) | No | - | qPCR, bulk, sorted cells |
27 | Female | 75 | Right ICA, CCA | 70 | No | No | No (macroscopic examination) | qPCR, bulk, sorted cells |
28 | Female | 68 | Left ICA | 68 | No | No | - | qPCR, sorted cells |
29 | Female | 60 | Left ICA | 70 | No | No | No (macroscopic examination) | qPCR, bulk |
30 | Female | 79 | Left ICA | 90 | No | Yes (2019) | No (macroscopic examination) | qPCR, sorted cells |
31 | Male | 62 | Right ICA, CCA, ECA | 80 | No | Yes (2019) | No (macroscopic examination) | qPCR, bulk, sorted cells |
32 | Male | 78 | Right ICA, CCA, ECA | 80 | No | No | Yes (macroscopic examination) | qPCR, bulk, sorted cells |
33 | Male | 68 | Left ICA, CCA | 60 | No | No | Yes (CT) | qPCR, bulk, sorted cells |
34 | Male | 57 | Right ICA | 70 | No | No | - | qPCR, bulk, sorted cells |
Cytokine | p.adj_NF1_IP-10 | p.adj_NF1_ TNF-α | p.adj_NF2 | p.adj_NF3 | p.adj_NF4 | p.adj_NF5 |
---|---|---|---|---|---|---|
IP-10 | - | 0.482 | 0.3931 | 0.1186 | 0.1262 | 0.1578 |
TNF-α | 0.1045 | - | 0.2502 | 0.0051 | 0.0005 | 0.6221 |
MDC | 0.0043 | 0.0311 | 0.0045 | 0.0051 | 0.0072 | 6.56 × 10−5 |
PDGF-AA | 0.008 | 0.0097 | 0.0036 | 0.048 | 0.0376 | 0.0001 |
MIP-1b | 9.54 × 10−7 | 3.15 × 10−5 | 2.38 × 10−6 | 1.19 × 10−6 | 1.19 × 10−6 | 1.19 × 10−6 |
Cytokine | Higher in Media | Higher in Plasma | Equal | p | p. adj |
---|---|---|---|---|---|
EGF | 8 | 10 | 5 | 0.8145 | 0.8145 |
FGF-2 | 10 | 3 | 10 | 0.0923 | 0.1203 |
Flt-3L | 13 | 5 | 5 | 0.0963 | 0.1203 |
Fractalkine | 8 | 4 | 11 | 0.3877 | 0.4308 |
G-CSF | 23 | 0 | 0 | 2.38 × 10−7 | 2.04 × 10−6 |
GM-CSF | 22 | 1 | 0 | 5.72 × 10−6 | 1.23× 10−5 |
GRO-α | 22 | 0 | 1 | 4.77 × 10−7 | 2.04 × 10−6 |
IFN-α2 | 9 | 0 | 14 | 0.0039 | 0.0062 |
IFN-γ | 8 | 1 | 14 | 0.0391 | 0.0558 |
IL-10 | 22 | 1 | 0 | 5.72 × 10−6 | 1.23 × 10−5 |
IL-12 (p40) | 9 | 0 | 14 | 0.0039 | 0.0062 |
IL-12 (p70) | 19 | 2 | 2 | 0.0002 | 0.0004 |
IL-13 | 14 | 3 | 6 | 0.0127 | 0.0191 |
IL-17A | 4 | 0 | 19 | 0.125 | 0.15 |
IL-1RA | 22 | 1 | 0 | 5.72 × 10−6 | 1.23× 10−5 |
IL-1α | 4 | 2 | 17 | 0.6875 | 0.7366 |
IL-1β | 23 | 0 | 0 | 2.38 × 10−7 | 2.04 × 10−6 |
IL-2 | 21 | 0 | 2 | 9.54 × 10−7 | 3.58 × 10−6 |
IL-3 | 0 | 3 | 20 | 0.25 | 0.2885 |
IL-4 | 20 | 2 | 1 | 0.0001 | 0.0002 |
IL-6 | 22 | 0 | 1 | 4.77 × 10−7 | 2.04 × 10−6 |
IL-7 | 23 | 0 | 0 | 2.38 × 10−7 | 2.04 × 10−6 |
IL-8 | 22 | 0 | 1 | 4.77 × 10−7 | 2.04 × 10−6 |
IL-9 | 10 | 3 | 10 | 0.0923 | 0.1203 |
MCP-1 | 22 | 0 | 1 | 4.77 × 10−7 | 2.04 × 10−6 |
MCP-3 | 22 | 1 | 0 | 5.72 × 10−6 | 1.23 × 10−5 |
MIP-1α | 22 | 1 | 0 | 5.72 × 10−6 | 1.23 × 10−5 |
TGF-α | 19 | 1 | 3 | 4.01 × 10−5 | 8.01 × 10−5 |
TNF-β | 7 | 5 | 11 | 0.7744 | 0.8011 |
VEGF | 22 | 1 | 0 | 5.72 × 10−6 | 1.23 × 10−5 |
Cytokine | Plasma Batch 1 LLOD—ULOD, pg/mL | Plasma Batch 2 LLOD—ULOD, pg/mL | Plasma Batch 3 LLOD—ULOD, pg/mL | Plasma Batch 4 LLOD—ULOD, pg/mL | Media Batch 1 LLOD—ULOD, pg/mL | Media Batch 2 LLOD—ULOD, pg/mL |
---|---|---|---|---|---|---|
PDGF-AA | 1.08–18,081.96 | 0.72–20,065.96 | 0.68–22,963.40 | 0.74–21,472.50 | 0.41–7948.93 | 0.39–13,017.89 |
PDGF-AB/BB | 4.62–20,000.74 | 5.96–20,000.56 | 13.50–20,005.30 | 4.68–20,006.54 | 2.30–10,000.65 | 2.19–9999.68 |
RANTES | 2.82–20,000.26 | 7.92–20,088.44 | 10.84–20,005.40 | 4.38–20,453.64 | 0.38–10,129.51 | 0.37–10,248.35 |
IL-1β | 0.70–19,923.88 | 0.90–19,250.36 | 0.36–20,150.88 | 0.64–20,332.98 | 0.39–10,443.04 | 0.30–11,237.48 |
IL-10 | 0.76–20,016.2 | 0.54–20,087.48 | 0.96–20,044.16 | 3.66–19,998.52 | 0.33–10,028.06 | 0.24–10,017.47 |
sCD40L | 13.48–19,999.94 | 18.50–20,000.02 | 4.12–19,999.88 | 13.96–20,002.84 | 10.07–9999.85 | 1.36–9987.78 |
MIP-1b | 0.22–12,393.88 | 0.34–12,831.10 | 4.34–3769.56 | 0.34–3032.96 | 0.27–4914.41 | 0.18–1692.67 |
IL-6 | 2.74–23,388.24 | 3.66–3997.56 | 3.40–23,807.74 | 0.12–3780.96 | 2.05–1988.68 | 2.45–1951.61 |
Fractalkine | 35.68–19,999.98 | 68.12–20,493.26 | 198.34–20,524.50 | 183.54–20,262.30 | 12.30–10,000.00 | 10.68–10,000.01 |
IL-8 | 0.84–19,924.34 | 0.82–16,925.78 | 0.30–21,787.96 | 0.62–21,842.58 | 0.38–7063.09 | 0.35–20,510.55 |
MCP-1 | 1.64–15,186.06 | 1.70–15,212.52 | 5.34–22,631.72 | 2.58–14,387.2 | 0.78–4628.32 | 0.97–6960.94 |
TNF-α | 0.92–20,004.12 | 0.50–20,101.88 | 3.54–20,084.66 | 0.54–20,071.08 | 0.25–10,341 | 1.96–10,139.21 |
IP-10 | 16.64–19,876.90 | 1.86–20,242.16 | 7.98–20,004.54 | 18.52–20,227.06 | 2.86–9755.18 | 4.51–10,272.06 |
IL-2 | 0.66–19,808.34 | 0.82–20,049.54 | 0.14–20,819.14 | 0.36–20,966.98 | 0.37–9673.02 | 0.35–10,523.19 |
MIP-1α | 3.08–7786.06 | 3.64–8746.82 | 3.74–8132.06 | 0.82–10,896.38 | 0.40–3145.19 | 2.30–3662.51 |
IL-3 | 0.80–19,592.42 | 0.78–19,410.88 | 0.72–19,399.46 | 0.78–24,308.44 | 0.39–9936.79 | 0.38–10,128.16 |
IL-4 | 23.30–21,478.88 | 21.70–20,690.38 | 44.64–20,970.98 | 2.18–20,447.08 | 8.34–10,217.39 | 7.04–10,061.89 |
IL-17A | 1.22–19,998.64 | 0.36–20,036.64 | 18.9–19,897.30 | 19.66–19,449.32 | 0.33–10,000.19 | 0.10–10,044.16 |
GM-CSF | 1.12–19,985.68 | 1.04–19,952.64 | 0.28–20,008.60 | 0.36–20,060.66 | 0.35–10,081.12 | 0.33–10,141.51 |
EGF | 16.62–28,290.20 | 4.78–23,766.96 | 5.26–22,773.60 | 4.04–31,188.30 | 1.49–10,956.76 | 2.53–20,522.10 |
FGF-2 | 19.96–19,338.72 | 32.42–20,007.40 | 109.66–19,240.40 | 44.70–19,212.38 | 5.71–9817.99 | 14.79–9878.73 |
Eotaxin | 3.74–19,556.24 | 4.22–19,079.68 | 106.86–19,986.64 | 8.86–20,118.48 | 2.04–8966.26 | 1.77–18,359.05 |
TGF-α | 0.78–3281.06 | 0.70–2056.56 | 0.74–1967.28 | 0.76–2872.28 | 0.36–40,674.65 | 0.38–2111.52 |
G-CSF | 15.84–19,999.92 | 2.18–19,999.72 | 28.78–19,999.82 | 5.02–19,947.50 | 0.55–9998.44 | 2.49–9027.19 |
Flt-3L | 3.72–19,999.18 | 1.58–20,078.86 | 9.48–20,069.72 | 2.92–20,223.54 | 1.05–9669.85 | 2.23–10,365.65 |
IFN-α2 | 19.16–19,999.40 | 5.32–19,999.80 | 12.38–20,003.94 | 5.40–19,787.18 | 2.07–10,011.73 | 1.82–10,080.39 |
IFN-γ | 0.96–19,977.34 | 0.68–19,995.24 | 3.20–21,083.48 | 28.84–19,624.38 | 0.53–9991.68 | 1.68–9999.95 |
GRO-α | 4.56–19,977.02 | 7.82–20,071.1 | 2.42–17,427.92 | 80.38–16,878.3 | 1.24–9433.38 | 3.52–11,568.69 |
MCP-3 | 9.5–18,631.18 | 6.34–19,245.76 | 56.72–20,128.68 | 88.14–20,728.5 | 4.14–7491.87 | 5.49–48,227.68 |
IL-12p40 | 4.68–20,027.44 | 3.90–20,000.00 | 6.68–20,001.66 | 9.84–19,992.42 | 2.12–10,056.59 | 2.00–10,037.33 |
MDC | 6.58–19,946.5 | 10.4–20,260.28 | 9.76–20,006.74 | 7.06–20,050.92 | 1.44–9986.42 | 1.83–10,090.48 |
IL-12p70 | 0.96–20,296.86 | 0.76–20,184.12 | 0.42–20,096.78 | 1.52–20,065.28 | 0.37–10,017.34 | 0.39–10,059.23 |
IL-13 | 0.86–20,059.92 | 0.48–20,062.56 | 4.52–20,116.04 | 3.40–20,084.60 | 0.29–10,067.40 | 1.78–10,045.41 |
IL-15 | 0.70–20,184.88 | 0.62–20,229.08 | 4.38–20,065.36 | 0.32–20,162.46 | 0.22–10,081.62 | 0.19–10,078.97 |
IL-1RA | 1.50–19,999.62 | 1.32–20,002.80 | 4.44–19,999.78 | 1.00–20,000.06 | 0.18–10,000.15 | 1.80–10,011.93 |
IL-1a | 3.96–19,998.02 | 1.18–19,997.60 | 3.86–20,019.58 | 3.38–20,099.74 | 0.44–9992.81 | 0.17–10,226.89 |
IL-9 | 0.8–19,708.56 | 0.62–20,592.08 | 0.62–20,388.62 | 1.88–20,125.58 | 0.36–10,155.81 | 0.28–10,849.89 |
IL-5 | 0.80–19,899.20 | 0.80–19,618.64 | 0.76–20,146.60 | 0.80–19,973.44 | 0.39–9542.06 | 0.39–10,085.75 |
IL-7 | 16.76–3983.94 | 3.30–2987.18 | 3.48–3961.70 | 2.28–3947.64 | 2.82–1991.33 | 2.20–1985.62 |
TNF-β | 3.46–19,999.18 | 0.56–20,000.48 | 4.54–19,998.36 | 0.16–19,655.16 | 0.27–9832.42 | 1.6–9714.92 |
VEGF | 20.68–20,226.02 | 1.06–20,301.96 | 97.94–20,089.88 | 13.24–20,053.22 | 10.55–10,443.48 | 12.00–10,430.18 |
Gene | Forward Primer | Reverse Primer | Probe |
---|---|---|---|
UBC | TTGGGTCGCAGTTCTTGTTTG | TGCCTTGACATTCTCGATGGT | VIC-TCGCTGTGATCGTCACTTGACAATG-BHQ2 |
MCP-1 (CCL2) | GTCTCTGCCGCCCTTCTG | GCGAGCCTCTGCACTGAG | VIC-ATAGCAGCCACCTTCATTCCCCAA-BHQ2 |
MIP1A (CCL3) | GGCTCTCTGCAACCAGTTCT | TTGGTTAGGAAGATGACACCG | VIC-AGATTCCACAGAATTTCATAGCTGACTAC-BHQ2 |
MIP1B (CCL4) | CCACCGCCTGCTGCTTTTC | CAGGATTCACTGGGATCAGC | VIC-AGCTTCCTCGCAACTTTGTGGTAGA-BHQ2 |
RANTES (CCL5) | TCATTGCTACTGCCCTCTGC | GTAGAAATACTCCTTGATGTGGG | ROX-CTGCTGCTTTGCCTACATTGCCC-BHQ2 |
FKN (CX3CL1) | CCATGTTCACCTACCAGAGC | ACACGGGCACCAGGACATA | ROX-GAAAGATGGCAGGAGAGATGGCG-BHQ2 |
Gene | Forward Primer | Reverse Primer |
---|---|---|
UBC | TTGGGTCGCAGTTCTTGTTTG | TGCCTTGACATTCTCGATGGT |
TNFA | CACTTTGGAGTGATCGGCCC | TTGTCACTCGGGGTTCGAGA |
IP-10 (CXCL10) | AAGTGGCATTCAAGGAGTACCT | GGACAAAATTGGCTTGCAGGA |
MDC (CCL22) | GCACTCCTGGTTGTCCTCG | GGCAGACGGTAACGGACG |
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Cytokine | Elevated in Plasma | Elevated in Medium | Criterion | Cytokine | Elevated in Plasma | Elevated in Medium | Criterion |
---|---|---|---|---|---|---|---|
EGF | No | No | Sign | IL-4 | No | Yes | Sign |
Eotaxin | Yes | No | Wilcoxon | IL-5 | No | No | Wilcoxon |
FGF-2 | No | No | Sign | IL-6 | No | Yes | Sign |
Flt-3L | No | No | Sign | IL-7 | No | Yes | Sign |
Fractalkine | No | No | Sign | IL-8 | No | Yes | Sign |
G-CSF | No | Yes | Sign | IL-9 | No | No | Sign |
GM-CSF | No | Yes | Sign | IP-10 | No | No | Wilcoxon |
GRO-α | No | Yes | Sign | MCP-1 | No | Yes | Sign |
IFN-α2 | No | Yes | Sign | MCP-3 | No | Yes | Sign |
IFN-γ | No | No | Sign | MDC | Yes | No | Wilcoxon |
IL-10 | No | Yes | Sign | MIP-1b | No | Yes | Wilcoxon |
IL-12p40 | No | Yes | Sign | MIP-1α | No | Yes | Sign |
IL-12p70 | No | Yes | Sign | PDGF-AA | Yes | No | Wilcoxon |
IL-13 | No | Yes | Sign | PDGF-AB/BB | Yes | No | Wilcoxon |
IL-15 | No | Yes | Wilcoxon | RANTES | Yes | No | Wilcoxon |
IL-17A | No | No | Sign | sCD40L | Yes | No | Wilcoxon |
IL-1α | No | No | Sign | TGF-α | No | Yes | Sign |
IL-1RA | No | Yes | Sign | TNF-α | No | No | Wilcoxon |
IL-1β | No | Yes | Sign | TNF-β | No | No | Sign |
IL-2 | No | Yes | Sign | VEGF | No | Yes | Sign |
IL-3 | No | No | Sign |
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Potashnikova, D.; Maryukhnich, E.; Vorobyeva, D.; Rusakovich, G.; Komissarov, A.; Tvorogova, A.; Gontarenko, V.; Vasilieva, E. Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy. Int. J. Mol. Sci. 2024, 25, 1030. https://doi.org/10.3390/ijms25021030
Potashnikova D, Maryukhnich E, Vorobyeva D, Rusakovich G, Komissarov A, Tvorogova A, Gontarenko V, Vasilieva E. Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy. International Journal of Molecular Sciences. 2024; 25(2):1030. https://doi.org/10.3390/ijms25021030
Chicago/Turabian StylePotashnikova, Daria, Elena Maryukhnich, Daria Vorobyeva, George Rusakovich, Alexey Komissarov, Anna Tvorogova, Vladimir Gontarenko, and Elena Vasilieva. 2024. "Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy" International Journal of Molecular Sciences 25, no. 2: 1030. https://doi.org/10.3390/ijms25021030