Role In Atherosclerotic Plaque Rupture Research Articles

The underlying molecular mechanisms and biomarkers of plaque vulnerability based on bioinformatics analysis

AimThe study aimed to identify the underlying differentially expressed genes (DEGs) and mechanism of unstable atherosclerotic plaque using bioinformatics methods.MethodsGSE120521, which includes four unstable samples and four stable atherosclerotic samples, was downloaded from the GEO database. DEGs were identified using LIMMA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed using the Database for metascape Visualization online tool. Based on the STRING database, protein–protein interactions (PPIs) network among DEGs were constructed. Regulatory networks were visualized using Cytoscape. We use the xCell to analyze the different immune cell subtypes.ResultsA total of 1626 DEGs (1034 up-regulated and 592 down-regulated DEGs) were identified between unstable and stable samples. I pulled 62 transcription factors (34 up-regulated TFs and 28 down-regulated TFs) from the Trust database. The up-regulated TFs were mainly enrichment in positive regulation of myeloid leukocyte differentiation, and the down-regulated TFs were mainly enrichment in connective tissue development. In the PPI network, RB1, CEBPA, PPARG, BATF was the most significantly up-regulated gene in ruptured atherosclerotic samples. The immune cell composition enriched in CD cells and macrophages in the unstable carotid plaque.ConclusionsUpregulated RB1, CEBPA, PPARG, BATF and down-regulated SRF, MYOCD, HEY2, GATA6 might perform critical promotional roles in atherosclerotic plaque rupture, furthermore, number and polarization of macrophages may play an important role in vulnerable plaques.

Plaque Structural Stress: Detection, Determinants and Role in Atherosclerotic Plaque Rupture and Progression.

Atherosclerosis remains a major cause of death worldwide, with most myocardial infarctions being due to rupture or erosion of coronary plaques. Although several imaging modalities can identify features that confer risk, major adverse cardiovascular event (MACE) rates attributable to each plaque are low, such that additional biomarkers are required to improve risk stratification at plaque and patient level. Coronary arteries are exposed to continual mechanical forces, and plaque rupture occurs when plaque structural stress (PSS) exceeds its mechanical strength. Prospective studies have shown that peak PSS is correlated with acute coronary syndrome (ACS) presentation, plaque rupture, and MACE, and provides additional prognostic information to imaging. In addition, PSS incorporates multiple variables, including plaque architecture, plaque material properties, and haemodynamic data into a defined solution, providing a more detailed overview of higher-risk lesions. We review the methods for calculation and determinants of PSS, imaging modalities used for modeling PSS, and idealized models that explore structural and geometric components that affect PSS. We also discuss current experimental and clinical data linking PSS to the natural history of coronary artery disease, and explore potential for refining treatment options and predicting future events.

Microvessels and glypican‐1 appears in atherosclerotic plaque in the aortic arch of aged rats

Rupture of atherosclerotic plaque is a major cause of death in acute cardiovascular and cerebrovascular events. We believe that the growth of plaques leads to microcirculation dysfunction, which promotes angiogenesis for establishing a new microcirculation in pathological tissues to achieve new homeostasis. However, rupture of atherosclerotic plaque occurs during the establishment of new microcirculation. There is abundant neovascularization in ruptured atherosclerotic plaques. It was demonstrated that endothelial glycocalyx is required for angiogenesis, but their specific angiogenic mechanism remains unclear. In order to investigate the relationship between endothelial glycocalyx and the angiogenesis of atherosclerotic vulnerable plaque, we detected the aortic arch plaque in 23-month-old rats and performed the immunofluorescence staining of CD31 and glypican-1 in frozen sections of the aortic root and aortic arch. Then, the role of glypican-1 in angiogenesis induced by fluid shear stress was analyzed by in vitro experiments. Results showed that there are abundant microvessels in the atherosclerotic plaque of the aortic arch in aged rats, and there is a high level of glypican-1 in the atherosclerotic plaque. In vitro experiments showed that the shedding of glypican-1 by phosphatidylinositol-specific phospholipase C (PI-PLC) could inhibit 25 dyn/cm2 shear stress-induced angiogenesis, indicating that glypican-1 plays an important role in angiogenesis in atherosclerotic plaque. The relationship between glypican-1 and neovascularization, and their roles in atherosclerotic plaque rupture need further study.

Investigation of the Underlying Genes and Mechanism of Macrophage-Enriched Ruptured Atherosclerotic Plaques Using Bioinformatics Method.

Aim: The study aimed to identify the underlying differentially expressed genes (DEGs) and mechanism of macrophage-enriched rupture atherosclerotic plaque using bioinformatics methods.Methods: GSE41571, which includes six stable samples and five ruptured atherosclerotic samples, was downloaded from the GEO database. After preprocessing, DEGs between ruptured and stable atherosclerotic samples were identified using LIMMA. Gene Ontology biological process (GO_BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of DEGs were performed using the Database for Annotation, Visualization, and Integration Discovery (DAVID) online tool. Based on the STRING database, protein-protein interactions (PPIs) network among DEGs were constructed. Regulatory relationships between miRNAs/transcriptional factors (TFs) and target genes were predicted using Enrichr, and regulatory networks were visualized using Cytoscape.Results: A total of 268 DEGs (64 up-regulated and 204 down-regulated DEGs) were identified between ruptured and stable samples. In the PPI network, collagen type III alpha 1 chain (COL3A1), collagen type I alpha 2 chain (COL1A2), and asporin (ASPN) were more than 15 interaction degrees. In the miRNA-target network, miR21 was highlighted with highest degrees and ASPN could be targeted by miR21. Functional enrichment analysis showed that COL3A1 and COL1A2 were significantly enriched in extracellular matrix organization and cell adhesion GO_BP terms. Pre-platelet basic protein (PPBP) was the most significantly up-regulated gene in ruptured atherosclerotic samples and enriched in immune response and inflammatory response GO_BP terms.Conclusions: Down-regulated COL3A1, COL1A2 and ASPN, and up-regulated PPBP might perform critical promotional roles in atherosclerotic plaque rupture. Furthermore, miR21 might be potential target to prevent atherosclerotic rupture.

Inhibition of EMMPRIN and MMP-9 Expression by Epigallocatechin-3-Gallate through 67-kDa Laminin Receptor in PMA-Induced Macrophages

Background/Aims: It is well documented that overexpression of EMMPRIN (extracellular matrix metalloproteinase inducer) and MMPs (matrix metalloproteinases) by monocytes/macrophages plays an important role in atherosclerotic plaque rupture. Green tea polyphenol epigallocatechin-3-gallate (EGCG) has a variety of pharmacological properties and exerts cardiovascular protective effects. Recently, the 67-kD laminin receptor (67LR) has been identified as a cell surface receptor of EGCG. The aim of the present study was to evaluate the effects of EGCG on the expression of EMMPRIN and MMP-9 in PMA-induced macrophages, and the potential mechanisms underlying its effects. Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Protein expression and MMP-9 activity were assayed by Western blot and Gelatin zymography, respectively. Real-time PCR was used to examine EMMPRIN and MMP-9 mRNA expression. Results: We showed that EGCG (10-50µmol/L) significantly inhibited the expression of EMMPRIN and MMP-9 and activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun N-terminal kinase (JNK) in PMA-induced macrophages. Downregulation of EMMPRIN by gene silencing hindered PMA-induced MMP-9 secretion and expression, indicating an important role of EMMPRIN in the inhibition of MMP-9 by EGCG. Moreover, 67LR was involved in EGCG-mediated suppression of EMMPRIN and MMP-9 expression. Anti-67LR antibody treatment led to abrogation of the inhibitory action of EGCG on the expression of EMMPRIN and MMP-9 and activation of ERK1/2, p38, and JNK. Conclusion: Our results indicate that EGCG restrains EMMPRIN and MMP-9 expression via 67LR in PMA-induced macrophages, which also suggests that EGCG may be a possible therapeutic agent for stabilizing atherosclerotic plaque.

Feasibility of [18F]-RGD for ex vivo imaging of atherosclerosis in detection of αvβ3 integrin expression

BackgroundInflammation and angiogenesis play an important role in atherosclerotic plaque rupture. Therefore, molecular imaging of these processes could be used for determination of rupture-prone atherosclerotic plaques. αvβ3 integrin is involved in the process of angiogenesis. Targeted imaging of αvβ3 integrin has been shown to be possible in previous studies on tumor models, using radiolabeled arginine-glycine-aspartate (RGD). Our aim was to investigate feasibility of ex vivo detection of αvβ3 integrin in carotid endarterectomy (CEA) specimens. Methods and ResultsNineteen CEA specimens were incubated in 5 MBq [18F]-RGD-K5 for 1 hour followed by 1 hour emission microPET scan. The results were quantified in 4 mm wide segments as percent incubation dose per gram (%Inc/g). Segmental-to-total ratio was calculated and presence of αvβ3 integrin and endothelial cells in each segment was confirmed by immunohistochemical staining for CD31 and αvβ3 integrin, respectively. [18F]-RGD-K5 uptake was heterogeneously distributed across CEA specimens and was localized within the vessel wall. Significant correlations were observed between segmental-to-total ratio with αvβ3 integrin staining score (r = 0.58, P = .038) and CD31 staining score (ρ = 0.67, P < .002). ConclusionThis study showed the feasibility of integrin imaging by determination of αvβ3 integrin expression in human atherosclerotic plaques.

The Inhibition of Oxidised Low-Density Lipoprotein-Induced Apoptosis of Macrophages by Recombinant Human Brain Natriuretic Peptide and the Underlying Mechanism

Objective: Macrophage apoptosis plays a key role in atherosclerotic plaque rupture. This study investigated the effects of recombinant human brain natriuretic peptide (BNP) on oxidised low-density lipoprotein (ox-LDL)-induced macrophage apoptosis and explored the underlying mechanism. Methods: A model of ox-LDL-induced macrophage injury was established to evaluate the role of BNP. Flow cytometry was employed to detect apoptosis and changes in mitochondrial membrane potential (ΔΨm), and confocal microscopy was used to determine cellular reactive oxygen species (ROS) levels. Additionally, reverse transcription-polymerase chain reaction and colourimetry were used to detect the mRNA expression and activity, respectively, of superoxide dismutase (SOD) and malondialdehyde (MDA). Results: Ox-LDL induced macrophage apoptosis in a concentration-dependent manner, and maximum apoptosis occurred at 100 μg/ml ox-LDL (45.62 ± 2.76 vs. 6.84 ± 1.94%; p < 0.05). Conversely, BNP suppressed macrophage apoptosis, with a maximal effect at 10^-9 mol/l (18.56 ± 1.79%; p < 0.05). Compared with the control group, intracellular ROS levels increased, ΔΨm decreased, SOD mRNA expression and activity decreased and MDA mRNA expression and content increased in the 100-μg/ml ox-LDL group (527.30 ± 36.20 vs. 100.00 ± 0.00%, 3.01 ± 0.52 vs. 9.67 ± 0.51%, 0.53 ± 0.18 vs. 1.00 ± 0.00, 256.6 ± 8.20 vs. 355.8 ± 9.58 U/ml, 1.59 ± 0.23 vs. 1.00 ± 0.00 and 29.4 ± 1.68 vs. 5.94 ± 0.51 nmol/ml; p < 0.05); these effects were significantly counteracted by 10^-9 mol/l BNP (237.30 ± 30.62%, 6.55 ± 1.57%, 0.90 ± 0.07, 310.4 ± 2.97 U/ml, 1.14 ± 0.10, 20.54 ± 1.55 nmol/ml; p < 0.05). Conclusion: BNP attenuates ox-LDL-induced macrophage apoptosis by suppressing oxidative stress and preventing ΔΨm loss.

Quantification of Inflammation Within Rabbit Atherosclerotic Plaques Using the Macrophage-Specific CT Contrast Agent N1177: A Comparison with 18F-FDG PET/CT and Histology

Macrophages play a key role in atherosclerotic plaque rupture. The iodine-based contrast agent N1177 accumulates in macrophages, allowing for their detection with CT. In this study, we tested whether the intensity of enhancement detected with CT in the aortic wall of rabbits injected with N1177 correlated with inflammatory activity evaluated with (18)F-FDG PET/CT and macrophage density on histology. Atherosclerotic plaques were induced in the aorta of New Zealand White rabbits (n = 7) by a repeated balloon injury (4 wk apart) and 4 mo of hyperlipemic diet. Noninjured rabbits, fed a chow diet, were used as controls (n = 3). A CT scan of the aorta (n = 10) was acquired in each rabbit before, during, and at 2 h after intravenous injection of N1177 (250 mg of iodine/kg). One week later, the same rabbits underwent PET/CT 3 h after injection of (18)F-FDG (37 MBq/kg [1 mCi/kg]). CT enhancement was calculated as the difference in aortic wall densities between images obtained before and images obtained at 2 h after injection of N1177. Mean standardized uptake values were measured on PET axial slices of the aorta in regions of interest encompassing the vessel wall. Macrophage density was measured by immunohistology (anti-RAM-11 antibody) on corresponding aortic cross-sections. N1177-enhanced CT measured stronger enhancement in the aortic wall of atherosclerotic rabbits than in control rabbits (10.0 +/- 5.2 vs. 2.0 +/- 2.1 Hounsfield units, respectively; P < 0.05). After the injection of (18)F-FDG, PET detected higher standardized uptake values in the aortic wall of atherosclerotic rabbits than in control rabbits (0.61 +/- 0.12 vs. 0.21 +/- 0.02; P < 0.05). The intensity of enhancement in the aortic wall measured with CT after injection of N1177 correlated with (18)F-FDG uptake on PET/CT (r = 0.61, P < 0.001) and macrophage density on immunohistology (r = 0.63, P < 0.001). The intensity of enhancement detected with CT in the aortic wall of rabbits injected with N1177 correlates with intense uptake of (18)F-FDG measured with PET and with macrophage density on histology, suggesting a role for N1177 in noninvasive identification of high-risk atherosclerotic plaques with CT.

Leukotriene Signaling in Atherosclerosis and Ischemia

The inflammatory process of atherosclerosis is associated with several pathophysiological reactions within the vascular wall. The arachidonic acid released by phospholipase A(2) serves as substrate for the production of a group of lipid mediators known as the leukotrienes, which induce pro-inflammatory signaling through activation of specific BLT and CysLT receptors. Leukotriene signaling has been implicated in early lipid retention and foam cell accumulation, as well as in the development of intimal hyperplasia and advanced atherosclerotic lesions. Furthermore, the association of leukotrienes with degradation of extracellular matrix has suggested a role in atherosclerotic plaque rupture. Finally, studies of either myocardial or cerebral ischemia and reperfusion indicate that leukotriene signaling in addition may be involved in the development of ischemic injury. Both leukotriene synthesis inhibitors and leukotriene receptor antagonists have been suggested to induce beneficial effects at different stages of the atherosclerosis process.

Interleukin 18 stimulates release of soluble lectin-like oxidized LDL receptor-1 (sLOX-1)

Lectin-like oxidized LDL receptor-1 (LOX-1) appears to play crucial roles in atherosclerotic plaque rupture. We previously reported that circulating soluble LOX-1 (sLOX-1) levels are elevated in acute coronary syndrome (ACS) and that sLOX-1 can be a specific and sensitive biomarker for ACS. A proinflammatory cytokine interleukin 18 (IL-18) and its receptor are prominently expressed in atherosclerotic plaques. In addition, circulating IL-18 levels were reported to be high in ACS. In this study, we have examined if IL-18 can stimulate shedding of LOX-1 and subsequent release of sLOX-1. After transfection with LOX-1 cDNA, HEK-293T cells were incubated with or without IL-18. Cell-conditioned media and total cell lysates were subjected to immunoblot analyses with an anti-LOX-1 monoclonal antibody. In addition, ADAM10 cDNA, ADAM10 siRNA or control vector were also co-transfected into HEK-293T cells, and the cell-conditioned media and total cell lysates were subjected to LOX-1 immunoblotting after treatment with or without IL-18. The cell-conditioned medium/total cell lysate ratios in the amounts of LOX-1 or sLOX-1 were determined as sLOX-1 cleavage ratios. IL-18 (10–100 ng/mL) stimulation increased the sLOX-1 cleavage by 3–4-fold in a concentration- and time-dependent manner. ADAM10 overexpression alone similarly enhanced the sLOX-1 cleavage. ADAM10 inhibition by ADAM10 siRNA transfection significantly suppressed IL-18-induced sLOX-1 cleavage. IL-18 similarly enhanced sLOX-1 cleavage in TNF-α-activated cultured endothelial cells, as well as LOX-1 transgenic mice in vivo. IL-18 appears one of the stimuli that enhance sLOX-1 release in ACS and ADAM10 may be involved in this process.

Elevated serum interleukin‐18 levels might reflect the high risk of hospitalization in patients on peritoneal dialysis

Interleukin (IL)-18 is a potent pro-inflammatory cytokine and plays a central role in atherosclerotic plaque rupture and accelerates atherosclerosis. The aim of this study was to determine serum IL-18 levels in patients on peritoneal dialysis (PD) and to assess their relationship with hospitalization. Forty-three PD patients and 20 healthy individuals were enrolled in this study. We investigated the relationship of the serum concentrations of IL-18 and other well-established atherosclerotic markers, such as asymmetric dimethylarginine (ADMA). Hospitalization data from over a 18-month period were prospectively obtained on all 43 PD patients. Classic factors were entered into a Cox regression model to predict first hospitalization. The serum levels of IL-18 in patients on PD were significantly higher than those of healthy individuals (228.5 +/- 140.3 pg/mL vs 154.8 +/- 44.7 pg/mL, P < 0.05, respectively). Furthermore, serum IL-18 levels showed a positive correlation with duration of PD, serum beta2 microglobulin and serum ADMA levels. Mean serum levels of IL-18 were significantly higher among patients who had experienced at least one hospitalization than those who had not (279.9 +/- 164.3 vs 158.5 +/- 43.9 pg/mL, P = 0.0426). Furthermore, the relative risk for first hospitalization for each increase in IL-18 (pg/mL) levels was associated with a 1.182 (95% confidence interval, 1.012-1.364; P = 0.0071) increase in the risk for future hospitalization events. The present study suggests the elevated serum IL-18 levels might increase the risk for future hospitalization in patients on PD.

Interaction between monocytes and vascular smooth muscle cells enhances matrix metalloproteinase-1 production.

Matrix metalloproteinase-1 (MMP-1) plays an important role in atherosclerotic plaque rupture. The purpose of this study was to investigate the expression of MMP-1 by cell-to-cell interactions between monocytes and vascular smooth muscle cells (VSMCs). Human VSMCs and THP-1 cells (human monocytoid cells) were cocultured. MMP-1 levels were measured by enzyme-linked immunosorbent assay. Collagenolytic activity was determined by fluorescent labeled-collagen digestion. Immunohistochemistry was performed to determine which types of cells produce MMP-1. Adding THP-1 cells to VSMCs markedly increased the MMP-1 levels and activity of the culture media. MMP-1 levels were maximal when the cellular ratio of THP-1 cells/VSMCs was 1.0. Immunohistochemistry revealed that both types of cells in the coculture produced MMP-1. Separated coculture experiments showed that both direct contact and a soluble factor(s) contributed to MMP-1 production. Neutralizing anti-interleukin (IL)-6 and tumor necrosis factor-alpha antibodies inhibited coculture conditioned medium-induced MMP-1 production by VSMCs and THP-1 cells. Protein kinase C inhibitors, tyrosine kinase inhibitors, and a mitogen-activated protein kinase inhibitor significantly inhibited MMP-1 production by cocultures. Direct cell-to-cell interaction between THP-1 cells and VSMCs enhanced MMP-1 synthesis in both types of cells. Increased local MMP-1 production and activity induced by monocyte-VSMC interaction play an important pathogenic role in atherosclerotic plaque rupture.