Search

Purpose: Foam cell macrophage (FCM) formation is an early event in atherosclerosis that could contribute to fibrous cap development, although it has also been strongly implicated in cap rupture and thrombus formation. To understand this apparent paradox we compared the transcriptome of FCM and non-foamy macrophages (NFM) produced in vivo using Illumina bead chips.Methods: Sponges were implanted into fat-fed ApoE null or chow-fed C57Bl6 mice to produce FCM or NFM, respectively. Cells were purified based on their buoyant density and/or differential adherence. Gene expression was examined using Ingenuity Pathway Analysis, GO annotation and clustering (DAVID Bioinformatics Resources). Data was confirmed using q-PCR and IHC.Results: The functions enriched/upregulated in FCM included connective tissue development, function and disorders as well as immune response, cell signalling. The primary canonical pathway upregulated in the FCM was liver X receptor/retinoid X receptor (LXR/RXR) activation, consistent with a recent study using peritoneal FCM from LDLr knockout mice (Spann et al Cell 2012;151:138). We confirmed that FCM had more mRNA for LXR, RXR and target genes using qRT-PCR, and used IHC to identify LXRα containing FCM in the sponges. GO identified hepatic fibrosis pathways as highly upregulated in the FCM and Ingenuity suggested a role for transforming growth factor β1 (TGFβ1). For example, there was an increase in mRNA for many extracellular matrix proteins (including collagens 1, 4, 6, 8, biglycan and decorin), connective tissue growth factor (CTGF), BMP-1 and Fos/FosB/Jun/JunB. FCM also had more mRNA for matrix proteases (including cathepsins C and E and matrix metalloproteinases 2 and 23), without a corresponding difference in their inhibitors. Using IHC we established that many cells within the sponges and brachiocephalic arteries from fat-fed ApoE mice contained CTGF. As TGFβ1-induced CTGF expression is known to be regulated via SMADs, we examined these tissues for the presence of phosphoSMAD2 (pSMAD2) by IHC and found that it was present in the cytoplasm and nucleus of sponge FCM and plaque cells.Conclusions: Our data confirm the paradox introduced by Spann and colleagues showing that FCMs are anti-inflammatory via LXR activation. We have extended this by showing that FCM can contribute to fibrosis and matrix deposition, possibly due to exposure to TGFβ1. Clearly additional factors, including mediators of innate and acquired immunity, must be responsible for converting FCM from an anti-inflammatory/pro-fibrotic phenotype into cells capable of mediating plaque rupture. [ABSTRACT FROM PUBLISHER]

Purpose: Classical and alternative macrophage activation could play important roles in atherosclerotic plaque progression and rupture, in part by increasing production of proteases, including matrix metalloproteinases (MMPs), which directly destabilize plaques. Lymphocyte-derived cytokines are believed essential for classical and alternative macrophage activation in plaques, although this hypothesis has not been rigorously tested.Methods and Results: We measured the expression of phenotypic markers and MMPs in classically and alternatively activated mouse macrophages in vitro. We then compared mRNA expression levels of these genes in foam cells derived from subcutaneous sponges of high fat fed ApoE knockout and ApoE/Rag-1 double knockout mice. Furthermore, we performed immunohistochemistry in aortic root and brachiocephalic arteries to detect marker and MMP protein expression in vivo. Classical activation of mouse macrophages in vitro (n=3-6) significantly increased NOS-2, COX-2 and MMPs-13 and -14, whereas alternative activation increased arginase-1, CD206, Ym-1 and MMP-19 expressions (p<0.05). Interestingly, LPS partially reversed the effect of IL-4 on Arg-1 and Ym-1 and completely reversed the overexpression of CD206, so that a combination of LPS and IL-4 generated a combined phenotype with markers of both classically and alternatively activated macrophages. Foam cells in subcutaneous sponges from ApoE mice also expressed phenotypic markers and MMPs, irrespective of Rag-1 genotype. The proportion of plaque staining for markers of classical or alternative activation were examined using immunohistochemistry in brachiocephalic arteries from mice that had been given a high-fat diet. Although the plaque size was smaller in male ApoE/Rag1 KO mice, little difference was found in the proportion of macrophages present (ApoE KO 29 ± 4.3% of plaque area (mean ± SEM); ApoE/Rag1 KO 33 ± 4.8%), or the levels of the markers of activation. For example, male mice had similar iNOS (ApoE KO 16 ± 4.3%; ApoE/Rag1 KO 19 ± 5.1%), arginase-1 (ApoE KO 13 ± 4.1%; ApoE/Rag1 KO 15 ± 4.0%) and Ym-1 content (ApoE KO 2.5 ± 0.80%; ApoE/Rag1 KO 8.7 ± 3.5%). These results suggest that half of the foamy macrophages in the plaques bore markers of classical or alternative activation, irrespective of genotype.Conclusions: Classical and alternative macrophage activation in sponge and plaque foam cell macrophages can occur independently of T- and B-lymphocytes. [ABSTRACT FROM AUTHOR]

Aims: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion., Methods and Results: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture., Conclusion: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion., Competing Interests: Conflict of interest: None of the authors have received any financial, personal, or professional support, other than the funding disclosed above that has a bearing on the data presented here. Dr Libby is an unpaid consultant to or involved in clinical trials for Amgen, AstraZeneca, Baim Institute, Beren Therapeutics, Esperion Therapeutics, Genentech, Kancera, Kowa Pharmaceuticals, Medimmune, Merck, Norvo Nordisk, Novartis, Pfizer, and Sanofi-Regeneron. Dr Libby is a member of the scientific advisory board for Amgen, Caristo Diagnostics, Cartesian Therapeutics, CSL Behring, DalCor Pharmaceuticals, Dewpoint Therapeutics, Kancera, Kowa Pharmaceuticals, Olatec Therapeutics, Medimmune, Novartis, PlaqueTec, TenSixteen Bio, and XBiotech, Inc. Dr Libby’s laboratory has received research funding in the last 2 years from Novartis. Dr Libby is on the Board of Directors of XBiotech, Inc. Dr Libby has a financial interest in XBiotech, a company developing therapeutic human antibodies. Dr Libby has a financial interest in TenSixteen Bio, a company targeting somatic mosaicism and clonal haematopoiesis of indeterminate potential (CHIP) to discover and develop novel therapeutics to treat age-related diseases. Dr Libby’s interests were reviewed and managed by Brigham and Women’s Hospital and Mass General Brigham in accordance with their conflict-of-interest policies., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Atherosclerosis underlies the predominant number of cardiovascular diseases and remains a leading cause of morbidity and mortality worldwide. The development, progression and formation of clinically relevant atherosclerotic plaques involves the interaction of distinct and over-lapping mechanisms which dictate the roles and actions of multiple resident and recruited cell types including endothelial cells, vascular smooth muscle cells, and monocyte/macrophages. The discovery of non-coding RNAs (ncRNAs) including microRNAs, long non-coding RNAs, and circular RNAs, and their identification as key mechanistic regulators of mRNA and protein expression has piqued interest in their potential contribution to atherosclerosis. Accruing evidence has revealed ncRNAs regulate pivotal cellular and molecular processes during all stages of atherosclerosis including cell invasion, growth, and survival; cellular uptake and efflux of lipids, expression and release of pro- and anti-inflammatory intermediaries, and proteolytic balance. The expression profile of ncRNAs within atherosclerotic lesions and the circulation have been determined with the aim of identifying individual or clusters of ncRNAs which may be viable therapeutic targets alongside deployment as biomarkers of atherosclerotic plaque progression. Consequently, numerous in vivo studies have been convened to determine the effects of moderating the function or expression of select ncRNAs in well-characterized animal models of atherosclerosis. Together, clinicopathological findings and studies in animal models have elucidated the multifaceted and frequently divergent effects ncRNAs impose both directly and indirectly on the formation and progression of atherosclerosis. From these findings' potential novel therapeutic targets and strategies have been discovered which may pave the way for further translational studies and possibly taken forward for clinical application., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Aims: MMPs contribute to atherosclerotic plaque progression and instability, but the relative potency of their endogenous tissue inhibitors of metalloproteinases (TIMPs) as protective factors has not been defined. We therefore investigated the impact of TIMP-1 and TIMP-2 knockout on atherosclerotic plaque burden and composition in apolipoprotein E-knockout (Apoe(-/-)) mice and studied the underlying effects on monocyte/macrophage behaviour., Methods and Results: Analysis of brachiocephalic artery plaques revealed comparable atherosclerotic lesion areas between TIMP-1(-/-) Apoe(-/-) or TIMP-2(-/-) Apoe(-/-) double deficient mice and relevant age-matched, strain-matched Apoe(-/-) controls after 8 weeks of high-fat feeding. However, lesions from TIMP-2(-/-) Apoe(-/-) mice had higher levels of markers associated with plaque vulnerability, including increased macrophage: vascular smooth muscle cell ratios, larger necrotic core areas, reduced collagen contents, increased macrophage proliferation, and apoptosis frequencies, compared with TIMP-1(-/-)Apoe(-/-) and controls. In contrast, TIMP-1(-/-) Apoe(-/-) animals only had a significant reduction in vascular smooth muscle cell content compared with Apoe(-/-) controls. In vitro and in vivo findings implicated heightened monocyte/macrophage invasion in the detrimental effects observed on atherosclerotic plaque composition in TIMP-2(-/-) Apoe(-/-) mice. Moreover, TIMP-2 specifically decreased MMP-14-dependent monocyte/macrophage infiltration into sites of experimentally induced inflammation and established atherosclerotic lesions., Conclusion: Our data demonstrate that TIMP-2 plays a greater protective role than TIMP-1 during the pathogenesis of atherosclerosis, in part by suppressing MMP-14-dependent monocyte/macrophage accumulation into plaques., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

After a period of relative senescence in the field of vascular smooth muscle cell (VSMC) research with particular regards to atherosclerosis, the last few years has witnessed a resurgence, with extensive research re-assessing potential molecular mechanisms and pathways that modulate VSMC behaviour within the atherosclerotic-prone vessel wall and the atherosclerotic plaque itself. Attention has focussed on the pathological contribution of VSMC in plaque calcification; systemic and local mediators such as inflammatory molecules and lipoproteins; autocrine and paracrine regulators which affect cell-cell and cell to matrix contacts alongside cytoskeletal changes. In this brief focused review, recent insights that have been gained into how a myriad of recently identified factors can influence the pathological behaviour of VSMC and their subsequent contribution to atherosclerotic plaque development and progression has been discussed. An overriding theme is the mechanisms involved in the alterations of VSMC function during atherosclerosis., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Aims: Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell-cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis., Methods and Results: Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (approximately 35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice., Conclusion: This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.

Objective: Matrix metalloproteinases (MMPs) form a large family of enzymes that collectively can degrade all components of the extracellular matrix, and there is widespread interest in developing MMP inhibitors for the prevention of atherosclerotic plaque rupture. We have therefore investigated the effects of a broad-spectrum MMP inhibitor, RS-130830, on plaque development and stability. This compound inhibits a wide range of MMPs at concentrations below 20 nmol/L., Methods: Apolipoprotein E knockout mice were fed a Western diet. Dietary administration of RS-130830 commenced at the same time as fat-feeding and continued for 8, 12, 26 or 36 weeks. To investigate the effect of RS-130830 on established plaques, mice were fed high-fat diet for 16 weeks before initiation of drug treatment and were terminated 20 weeks after this., Results: Broad-spectrum MMP inhibition was associated with a significant increase in plaque area, but there was no change in the incidence of plaque rupture. There were unfavourable changes in phenotypic characteristics associated with plaque instability, such as an increased lipid content and decreased collagen content., Conclusions: These data suggest that broad-spectrum MMP inhibition RS-130830 does not have a beneficial effect on atherosclerosis in the apolipoprotein E knockout mouse model, and indicate that more selective compounds would be preferable.

The relationship between degradation of basement membranes, metalloproteinase (MMP) activity and smooth muscle cell (SMC) migration and proliferation has not been previously investigated in any intervention study. We used adenoviral overexpression of tissue inhibitors of metalloproteinases (TIMPs) in cultured human saphenous veins. By immunocytochemistry, the percentage of medial SMC surrounded by basement membrane type IV collagen (Coll-IV) decreased from 93+/-1 to 77+/-4% and 82+/-1% (n=18, both P<0.001) after 7 and 14 days of culture, respectively, while all SMC that migrated to the neointima lacked Coll-IV. Overexpression of TIMP-1 or TIMP-3 significantly increased the percentage of medial SMC surrounded by Coll-IV (94+/-2 or 98+/-2%, respectively, both P<0.01 vs. no treatment) and decreased the number of neointimal SMC. Some 44+/-18 and 30+/-6%, respectively, of BrdU or PCNA labeled medial SMC remained surrounded by type IV collagen and this was not affected by overexpression of TIMP-1 or TIMP-3. We conclude that MMPs mediate loss of basement membrane and this is closely related to SMC migration. SMC proliferation does not require complete basement membrane degradation, which itself does not require MMPs in proliferating SMC.