Objective To determine the relationship between increased triggering receptor expressed on myeloid cells (TREM)-1 and plaque stability in atherosclerotic carotid stenosis. Methods The mRNA transcripts and protein for TREM-1, MMP-1, MMP-9, collagen type I (COL1A1) and collagen type III (COL3A1) were analyzed by qPCR and immunofluorescence in both tissues and VSMCs isolated from atherosclerotic carotid plaques of symptomatic and asymptomatic patients with carotid stenosis. Results The TREM-1, MMP-1 and MMP-9 mRNA transcripts were significantly increased (TREM-1, p<0.01; MMP-1, p<0.01 and MMP-9, p<0.001) while COL1A1 and COL3A1 mRNA transcripts were decreased (p<0.001) in VSMCs isolated from carotid plaques of symptomatic (S) than asymptomatic (AS) patients. Stimulation of cells with TNF-α further increased the mRNA transcripts of TREM-1, MMPs, COL1A1 and COL3A1. Modulation of TREM-1 by treatment with TREM-1 decoy receptor rTREM-1/Fc, and either TREM-1 antibodies or TREM-1 siRNA attenuated the TNF-α induced expression of MMP-1 and MMP-9 (p<0.01) and COL1A1 and COL3A1 (p<0.01) in S compared to AS VSMCs isolated from carotid plaques. Inhibition of NF-kB (BAY 11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways decreased the expression of TREM-1 (p<0.01), MMP-1 (p<0.001) and MMP-9 (p<0.01) in TNF-α treated VSMCs isolated from S carotid plaques compared to AS patients. Conclusion Increased expression of TREM-1 in S compared to AS patients involving MMP-1 and MMP-9 suggest a potential role of TREM-1 in plaque destabilization. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.
The data described herein are related to the article entitled “Tumor necrosis factor-α regulates triggering receptor expressed on myeloid cells-1-dependent matrix metalloproteinases in the carotid plaques of symptomatic patients with carotid stenosis” (Rao et al., 2016) [1]. Additional data are provided on the dose–response effect of TNF-α, TREM-1 antibody and recombinant rTREM-1/Fc fusion chimera (TREM-1/FC) on the expression of MMP-1 and MMP-9 in vascular smooth muscle cells (VSMCs) isolated from human carotid endarterectomy tissues. Data are also presented on the distribution of CD86+ M1- and CD206+ M2-macrophages and their co-localization with TREM-1 in symptomatic carotid plaques as visualized by dual immunofluorescence. The interpretation of this data and further extensive insights can be found in Rao et al. (2016) [1].
Rao VH, Rai V, Stoupa S, Agrawal DK. Blockade of Ets-1 attenuates epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells. Am J Physiol Heart Circ Physiol 309: H1075-H1086, 2015. First published August 7, 2015 doi:10.1152/ajpheart.00378.2015.-Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (␣ 1) and collagen III (␣1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways. carotid plaque; collagen types I and III; epidermal growth factor; matrix metalloproteinases; v-ets erythroblastosis virus E26 oncogene homologue 1; vascular smooth muscle cells NEW & NOTEWORTHY This study provides a mechanistic insight into the role of Ets-1 regulated EGF induced collagen loss involving p38-MAPK and JNK signaling pathways in human carotid plaques with carotid stenosis. Selective blockade of Ets-1 and EGF receptor may be a novel strategy and promising target for treating unstable and vulnerable plaques.ATHEROSCLEROSIS IS A COMPLEX disease with coronary thrombus leading to stroke and is the major cause of morbidity and mortality throughout the world (8, 24). The matrix accumulation and degradation in the extracellular matrix (ECM) may determine the outcome of plaque stability (1). Proteases produced by the cellular components of the plaque are thought to be mainly responsible for thinning of the plaque cap and the development of myocardial infarction and stroke. Inflammatory cytokines (tumor necrosis factor-␣ and interleukin-1) and growth factors [epidermal growth factor ...
Single particle electron cryo-microscopy (cryo-EM) of RNA alone has not been studied at high resolution as in the case of RNA-protein complexes, because of the intrinsic conformational variability that presents great challenges in particle classification for high resolution structure determination. T-box riboswitches interact with specific tRNAs and sense the corresponding amino acid levels in Gram-positive bacteria. The complete structure of the T-box/tRNA (80 kDa) complex remains unknown. We here report a subnanometer cryo-EM map of this RNA complex, in which major and minor grooves are unambiguously resolved. This result differs from the two small angle X-ray scattering (SAXS) models reported recently, and illustrates the interaction interface between tRNA and T-box riboswitch.
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