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Cited by 55Cerebrovascular and Carotid Artery DiseasesProtease and Inhibitor MechanismsAtherosclerosis and Cardiovascular Diseases

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Distribution of Matrix Metalloproteinases in Human Atherosclerotic Carotid Plaques and Their Production by Smooth Muscle Cells and Macrophage Subsets

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Jager, Nynke A., Wallis de Vries, Bastiaan M., Hillebrands, Jan-Luuk, Harlaar, Niels J., Tio, René A., Slart, Riemer H. J. A., van Dam, Gooitzen M., Boersma, Hendrikus H., Zeebregts, Clark J., Westra, Johanna·2016·Molecular Imaging and Biology
DOI 10.1007/s11307-015-0882-0PMC PMC4783451
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Abstract

PURPOSE: In this study, the potential of matrix metalloproteinase (MMP) sense for detection of atherosclerotic plaque instability was explored. Secondly, expression of MMPs by macrophage subtypes and smooth muscle cells (SMCs) was investigated. PROCEDURES: Twenty-three consecutive plaques removed during carotid endarterectomy were incubated in MMPSense™ 680 and imaged with IVIS® Spectrum. mRNA levels of MMPs, macrophage markers, and SMCs were determined in plaque specimens, and in in vitro differentiated M1 and M2 macrophages. RESULTS: There was a significant difference between autofluorescence signals and MMPSense signals, both on the intraluminal and extraluminal sides of plaques. MMP-9 and CD68 messenger RNA (mRNA) expression was higher in hot spots, whereas MMP-2 and αSMA expression was higher in cold spots. In vitro M2 macrophages had higher mRNA expression of MMP-1, MMP-9, MMP-12, and TIMP-1 compared to M1 macrophages. CONCLUSION: MMP-9 is most dominantly MMP present in atherosclerotic plaques and is produced by M2 rather than M1 macrophages.

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Fig. 1. Ex vivo fluorescence imaging of human atherosclerotic plaques. Fluorescence signals reflecting MMPSense activation in specific areas of plaque intraluminally (upper) and extraluminally (bottom): incubation in phosphate-buffered saline (left), autofluorescence (middle), and incubation with MMPSense.
Fig. 1. Ex vivo fluorescence imaging of human atherosclerotic plaques. Fluorescence signals reflecting MMPSense activation in specific areas of plaque intraluminally (upper) and extraluminally (bottom): incubation in phosphate-buffered saline (left), autofluorescence (middle), and incubation with MMPSense.CC BY · PubMed Central
Fig. 2. Intensity of fluorescence signals after incubation with MMPSense compared to autofluorescence. Fluorescence signals of plaques incubated with MMPSense (MMP) were increased both a intraluminally and b extraluminally when compared with autofluorescence (AF). Intraluminal (IL) side (mean value 6.34 × 109 vs 1.09 × 109; p < 0.0001) and extraluminal (EL) side (mean value 6.12 × 109 vs 1.04 × 109; p < 0.0001).
Fig. 2. Intensity of fluorescence signals after incubation with MMPSense compared to autofluorescence. Fluorescence signals of plaques incubated with MMPSense (MMP) were increased both a intraluminally and b extraluminally when compared with autofluorescence (AF). Intraluminal (IL) side (mean value 6.34 × 109 vs 1.09 × 109; p < 0.0001) and extraluminal (EL) side (mean value 6.12 × 109 vs 1.04 × 109; p < 0.0001).CC BY · PubMed Central
Fig. 3. Expression of MMPs in atherosclerotic plaques. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to determine relative mRNA expression of different genes in human atherosclerotic plaques. a Areas with high intensity (hot spots) and low intensity (cold spots) determined after incubation with MMPSense were excised from four imaged plaques and subjected to RNA isolation and qRT-PCR. mRNA of MMP-2, MMP-9, CD68, and αSMA, expressed as fold change in hot spots versus cold spots, was determined. b Relative mRNA expression of MMP-2, MMP-9, αSMA, and CD68 was determined in slices from four unimaged plaques with qRT-PCR. MMP-9 and CD68 mRNA expression was significantly correlated and also MMP-2 and αSMA mRNA expression (Spearman’s correlation). c Relative expression levels of different MMPs were determined with qRT-PCR in slices from four unimaged plaques. d Immunohistochemical staining of CD68 (middle panel) and MMP9 (right panel) in a plaque specimen (left panel).
Fig. 3. Expression of MMPs in atherosclerotic plaques. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to determine relative mRNA expression of different genes in human atherosclerotic plaques. a Areas with high intensity (hot spots) and low intensity (cold spots) determined after incubation with MMPSense were excised from four imaged plaques and subjected to RNA isolation and qRT-PCR. mRNA of MMP-2, MMP-9, CD68, and αSMA, expressed as fold change in hot spots versus cold spots, was determined. b Relative mRNA expression of MMP-2, MMP-9, αSMA, and CD68 was determined in slices from four unimaged plaques with qRT-PCR. MMP-9 and CD68 mRNA expression was significantly correlated and also MMP-2 and αSMA mRNA expression (Spearman’s correlation). c Relative expression levels of different MMPs were determined with qRT-PCR in slices from four unimaged plaques. d Immunohistochemical staining of CD68 (middle panel) and MMP9 (right panel) in a plaque specimen (left panel).CC BY · PubMed Central
Fig. 4. Relative expression of MMP mRNA levels in macrophages from ten healthy volunteers and in SMCs. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers and subjected to RNA isolation and qRT-PCR. Significantly higher relative expression in M2-like macrophages (gray bars) compared to M1-like macrophages (open bars) was measured for a MMP-1, b TIMP-1, and d MMP-12. c MMP-9 was also higher. Relative expression of MMP-1 and TIMP-1 in SMCs is added in panels a and b, expression of MMP-9 and MMP-12 was undetectable in SMCs. Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05, **p < 0.01.
Fig. 4. Relative expression of MMP mRNA levels in macrophages from ten healthy volunteers and in SMCs. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers and subjected to RNA isolation and qRT-PCR. Significantly higher relative expression in M2-like macrophages (gray bars) compared to M1-like macrophages (open bars) was measured for a MMP-1, b TIMP-1, and d MMP-12. c MMP-9 was also higher. Relative expression of MMP-1 and TIMP-1 in SMCs is added in panels a and b, expression of MMP-9 and MMP-12 was undetectable in SMCs. Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05, **p < 0.01.CC BY · PubMed Central
Fig. 5. Relative expression of MMP mRNA levels in macrophages from ten healthy volunteers and in SMCs. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers and subjected to RNA isolation and qRT-PCR. Significantly lower relative expression in M2 macrophages (gray bars) compared to M1-like macrophages (open bars) was measured for a MMP-2 and b MMP-14. Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05, **p < 0.01.
Fig. 5. Relative expression of MMP mRNA levels in macrophages from ten healthy volunteers and in SMCs. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers and subjected to RNA isolation and qRT-PCR. Significantly lower relative expression in M2 macrophages (gray bars) compared to M1-like macrophages (open bars) was measured for a MMP-2 and b MMP-14. Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05, **p < 0.01.CC BY · PubMed Central
Fig. 6. MMP-9 protein levels in supernatants of cultured M1 and M2 macrophages measured by ELISA. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers, and supernatant was harvested after differentiation. MMP-9 protein levels were measured by ELISA, and these were higher in M2 macrophages (gray bars) compared to M1 macrophages (open bars). Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05.
Fig. 6. MMP-9 protein levels in supernatants of cultured M1 and M2 macrophages measured by ELISA. M1- and M2-polarized macrophages were cultured from monocytes from ten healthy volunteers, and supernatant was harvested after differentiation. MMP-9 protein levels were measured by ELISA, and these were higher in M2 macrophages (gray bars) compared to M1 macrophages (open bars). Significant differences (Wilcoxon paired t test) of M2 subtype compared to M1 macrophages is shown in figure: *p < 0.05.CC BY · PubMed Central

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Research works printed but not linked: 32

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Metadata

Authors
Jager, N. A., Wallis de Vries, B. M., Hillebrands, J.-L., Harlaar, N. J., Tio, R. A., Slart, R. H. J. A., van Dam, G. M., Boersma, H. H., Zeebregts, C. J., & Westra, J.
Affiliations
University Medical Center Groningen (NL)
Corresponding author
Zeebregts, Clark J.
ORCID
Venue
Molecular Imaging and Biology
Publisher
Springer Science and Business Media LLC
Published
2015-09-16
Volume
18
Issue
2
Pages
283-291
ISSN
  • 1536-1632 (ISSN-L)
  • 1860-2002
DOI
10.1007/s11307-015-0882-0
PMC
PMC4783451
OpenAlex
W2173658293
Cited by
55
References
1
Citations by year
2026: 6 · 2025: 3 · 2024: 3 · 2023: 12 · 2022: 3 · 2021: 7 · 2020: 6 · 2019: 4 · 2018: 5 · 2017: 2 · 2016: 4
FWCI
2.90
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Imaging and Biology (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (publishedVersion, cc-by)
  • University of Groningen research database (University of Groningen / Centre for Information Technology) (submittedVersion, cc-by)
Topics
  • Health Sciences › Medicine › Cardiology and Cardiovascular Medicine › Cerebrovascular and Carotid Artery Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Life Sciences › Immunology and Microbiology › Immunology › Atherosclerosis and Cardiovascular Diseases
Keywords
matrix metalloproteinases, atherosclerotic carotid plaque, plaque vulnerability, MMP-9, macrophage polarization, M1 macrophages, M2 macrophages, smooth muscle cells, autofluorescence, CD68, alpha-smooth muscle actin, MMP-2, MMP-12, TIMP-1, carotid endarterectomy
MeSH terms
Aged, Aged, 80 and over, Demography, Enzyme-Linked Immunosorbent Assay, Female, Fluorescence, Humans, Macrophages, Male, Middle Aged, Risk Factors, RNA, Messenger, Gene Expression Regulation, Enzymologic, Carotid Stenosis, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, Myocytes, Smooth Muscle, Plaque, Atherosclerotic

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