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.

Aims: Unstable atherosclerotic plaques in carotid artery are characterized by rupture of their fibrous cap, leading to transient ischemic attack or stroke. However, the underlying mechanisms are unclear. Our aim was to investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) on plaque stability involving matrix metalloproteinases (MMPs) and examine the effect of inflammatory cytokine TNF-α in symptomatic (S) compared to asymptomatic (AS) patients with carotid stenosis. Methods and Results: The mRNA transcripts for TREM-1, MMPs (MMP-1, MMP-3, and MMP-9) and collagen type I and III were analyzed by qPCR and immunofluorescence, respectively in whole plaque and plaque smooth muscle cells (pSMCs) isolated from carotid endarterectomy tissues of patients with carotid stenosis. The mRNA transcripts of TREM-1, MMP-1 and MMP-9 were increased while Col I and Col III mRNA transcripts were decreased in pSMCs of S compared to AS patients. Stimulation of pSMCs with TNF-α further increased the mRNA transcripts of TREM-1 and MMPs without any additional effect on Col I (α1) or Col III (α1) mRNA levels. The inhibitors of NF-kB (BAY11-7085), JNK (SP600125) and PI3K (LY294002) signaling pathways attenuated the effect of TNF-α. Modulation of TREM-1 in TNF-α-treated pSMCs by the administration of TREM-1 decoy receptor rTREM-1/Fc, monoclonal antibodies, or siRNA to TREM-1 attenuated the expression of MMPs and Col I (α1) and Col III (α1) in both S and AS groups. Conclusion: Increased expression of TREM-1 in pSMCs of symptomatic than in asymptomatic patients suggest a potential role of TREM-1 in plaque destabilization. This is further supported by the pronounced effect of inflammatory cytokine, TNF-α, to increase of mRNA transcripts for TREM-1 and MMPs. Selective blockade of TREM-1 may contribute to the development of new therapies and promising targets for stabilizing vulnerable atherosclerotic plaques.

Introduction: Coronary thrombosis and rupture of plaques leading to stroke are major causes of morbidity and mortality. Here, we examined the role of v-ets erythroblastosis virus E26 oncogene homologue-1 (ETS-1) on the epidermal growth factor (EGF)-induced destabilization of collagen type I (Col-Iα1) and collagen type III (Col-IIIα1) involving matrix metalloproteinase-1 (MMP-1) and MMP-9 in isolated vascular smooth muscle cells (VSMCs) from the plaques of symptomatic (S) and asymptomatic (AS) patients with carotid stenosis. To understand the molecular mechanisms underlying EGFR-regulated ETS-1, the signaling pathways directing ETS-1 production was investigated and examined if EGF-induced loss of fibrillar collagens in patients with carotid stenosis is regulated by ETS-1. Methods: VSMCs isolated from carotid plaques of both AS and S patients were treated with or without EGF. The mRNA transcripts and protein for ETS-1, MMP-9 and -1, EGFR, Col-I(α1) and Col-III(α1) were analyzed by qPCR and immunofluorescence, respectively. Results: Treatment of VSMCs with EGF significantly increased ETS-1 expression and immunoreactivity in S compared to AS. EGFR inhibitor (AG1478) decreased ETS-1 expression and upregulated Col-I(α1) and Col-III(α1) in EGF-treated VSMCs of both groups. In the transfection experiments, overexpression of ETS-1 in VSMCs increased mRNA transcripts of MMP-9 and MMP-1 and decreased the collagen mRNA transcripts. Knock-down of ETS-1 gene with either siRNA or transfection with dominant-negative form (ETS-DN) blocked EGF-induced MMP-1 and MMP-9 expression and increased mRNA transcripts for Col-I(α1) and Col-III(α1) in S compared to AS group. Inhibitors of p38MAPK (SB202190) and JNK-II (SP600125) decreased ETS-1, MMP-1 and MMP-9 transcripts and increased collagen transcripts in EGF-treated VSMCs, suggesting that p38MAPK and JNK pathways are involved in EGFR-induced collagen loss. Conclusion: These findings revealed a novel mechanism of ETS-1-regulated EGF-induced collagen loss in human carotid plaques and this could be a leading cause of plaque instability in patients with carotid stenosis. Selective blockade of ETS-1 and EGFR may be novel strategy and promising target for treating unstable and vulnerable plaques.

Small-molecule aptamers are composed of RNA or DNA sequences with intricate secondary and tertiary structure allowing them to perform many functions, including ligand binding and catalytic events.