Low Levels of CD4+CD28null T Cells at Baseline Are Associated With First-Time Coronary Events in a Prospective Population-Based Case-Control Cohort.

OBJECTIVE: CD4+CD28null T cells have been shown to be associated with recurrent coronary events and suggested as potential biomarker and therapeutic target. It is unknown whether CD4+CD28null T cells associate with first-time cardiovascular events. We examined CD4+CD28null T cells in a prospective population-based cohort and in patients with advanced atherosclerosis. Approach and Results: CD4+CD28null T cells were quantified in 272 individuals experiencing a first-time coronary event during up to 17 years of follow-up and 272 age- and sex-matched controls in a case-control study, nested within the population-based Malmö Diet and Cancer study. The highest tertile of CD4+CD28null T cells was associated with a lower incidence of first-time coronary events compared with the lowest tertile (odds ratio, 0.48 [95% CI, 0.29-0.79], P=0.004) when adjusting for Framingham risk factors. This association remained significant for events recorded after >9 years of follow-up, when most coronary events occurred, but not during the first 9 years of follow-up, despite similar odds ratio. Additionally, we analyzed CD4+CD28null T cells in 201 patients with advanced atherosclerosis undergoing carotid endarterectomy. The adjusted hazard ratio for cardiovascular events in patients with advanced atherosclerosis was 2.11 (95% CI, 1.10-4.05, P=0.024), comparing the highest with the lowest CD4+CD28null T-cell tertile. CONCLUSIONS: Our findings reveal complex associations between CD4+CD28null T cells and cardiovascular disease. Although we confirm the reported positive associations with an adverse prognosis in patients with already established disease, the opposite associations with first-time coronary events in the population-based cohort may limit the clinical use of CD4+CD28null T cells.

Reduced oxidized LDL in T2D plaques is associated with a greater statin usage but not with future cardiovascular events.

BACKGROUND: Type 2 diabetes (T2D) patients are at a greater risk of cardiovascular events due to aggravated atherosclerosis. Oxidized LDL (oxLDL) has been shown to be increased in T2D plaques and suggested to contribute to plaque ruptures. Despite intensified statin treatment during the last decade the higher risk for events remains. Here, we explored if intensified statin treatment was associated with reduced oxLDL in T2D plaques and if oxLDL predicts cardiovascular events, to elucidate whether further plaque oxLDL reduction would be a promising therapeutic target. METHODS: Carotid plaque OxLDL levels and plasma lipoproteins were assessed in 200 patients. Plaque oxLDL was located by immunohistochemistry. Plaque cytokines, cells and scavenger receptor gene expression were quantified by Luminex, immunohistochemistry and RNA sequencing, respectively. Clinical information and events during follow-up were obtained from national registers. RESULTS: Plaque oxLDL levels correlated with markers of inflammatory activity, endothelial activation and plasma LDL cholesterol (r = 0.22-0.32 and p ≤ 0.01 for all). T2D individuals exhibited lower plaque levels of oxLDL, sLOX-1(a marker of endothelial activation) and plasma LDL cholesterol (p = 0.001, p = 0.006 and p = 0.009). No increased gene expression of scavenger receptors was identified in T2D plaques. The lower oxLDL content in T2D plaques was associated with a greater statin usage (p = 0.026). Supporting this, a linear regression model showed that statin treatment was the factor with the strongest association to plaque oxLDL and plasma LDL cholesterol (p < 0.001 for both). However, patients with T2D more frequently suffered from symptoms and yet plaque levels of oxLDL did not predict cardiovascular events in T2D (findings are summarized in Fig. 1a). CONCLUSIONS: This study points out the importance of statin treatment in affecting plaque biology in T2D. It also implies that other biological components, beyond oxLDL, need to be identified and targeted to further reduce the risk of events among T2D patients receiving statin treatment.

sTRAIL-R2 (Soluble TNF [Tumor Necrosis Factor]-Related Apoptosis-Inducing Ligand Receptor 2) a Marker of Plaque Cell Apoptosis and Cardiovascular Events.

Background and Purpose- Cellular apoptosis is an important feature in atherosclerosis, contributing to necrotic core formation, and plaque vulnerability. Activation of the death receptor TRAIL-R2 (TNF [tumor necrosis factor]-related apoptosis-inducing ligand receptor 2) through its ligand tumor necrosis factor-relate apoptosis-inducing ligand (TRAIL), induces apoptosis in cells in vitro. sTRAIL-R2 (soluble TRAIL-R2) was recently shown to predict cardiovascular events in healthy individuals. In the present study, we explored if plaque levels of sTRAIL-R2 and sTRAIL reflect plaque apoptosis and vulnerability and if plasma levels of these markers predict future events in subjects with advanced atherosclerosis. Methods- Plasma from 558 patients and 202 carotid plaques from the Carotid Plaque Imaging Project biobank were used. sTRAIL-R2, sTRAIL, and caspase-8 levels were assessed using a Proseek Multiplex CVD96×96 assay. Active caspase-3 was measured using ELISA to assess plaque apoptosis. Plaque morphology was studied by immunohistochemistry. Inflammatory cytokines were assessed by Luminex. mRNA levels were quantified by RNA sequencing. Monocytes, T cells, B cells, and human coronary artery smooth muscle cells were used to study sTRAIL-R2 and sTRAIL release on cell apoptosis and inflammatory stimuli in vitro. Results- Plaque levels of sTRAIL-R2 and sTRAIL correlated to markers of extrinsic induced apoptosis (caspase-3 and -8). sTRAIL-R2 and sTRAIL protein expression were increased in symptomatic carotid plaques and patients with higher plasma levels of sTRAIL-R2 had a higher risk of future cardiovascular events. sTRAIL-R2 and sTRAIL were released upon activation of the extrinsic apoptosis pathway in vitro. sTRAIL-R2 and sTRAIL correlated with inflammatory cytokines, to CD68 expression and inversely to α-actin in the plaque tissue. Conclusions- The present study shows that sTRAIL-R2 and sTRAIL are associated to human plaque cell apoptosis, plaque inflammatory activity, and with symptomatic carotid plaques. Furthermore, high plasma levels of sTRAIL-R2 in plasma predict, independently, future cardiovascular events in individuals with manifest atherosclerotic disease.

Transforming growth factor-ß2 is associated with atherosclerotic plaque stability and lower risk for cardiovascular events.

AIMS: Transforming growth factor-beta (TGF-ß) exists in three isoforms TGF-ß1, -ß2, and -ß3. TGF-ß1 has been suggested to be important for maintaining plaque stability, yet the role of TGF-ß2 and -ß3 in atherosclerosis remains to be investigated.This study explores the association of the three isoforms of TGF-ß with plaque stability in the human atherosclerotic disease. METHODS AND RESULTS: TGF-ß1, -ß2, and -ß3 proteins were quantified in 223 human carotid plaques by immunoassays. Indications for the endarterectomy were: symptomatic carotid plaque with stenosis >70% or without symptoms and >80% stenosis. Plaque mRNA levels were assessed by RNA sequencing. Plaque components and extracellular matrix were measured histologically and biochemically. Matrix metalloproteinases and monocyte chemoattractant protein-1 (MCP-1) was measured with immunoassays. The effect of TGF-ß2 on inflammation and protease activity was investigated in vitro using THP-1 and RAW264.7 macrophages. Patients were followed longitudinally for cardiovascular (CV) events.TGF-ß2 was the most abundant isoform and was increased at both protein and mRNA levels in asymptomatic plaques. TGF-ß2 was the main determinant separating asymptomatic plaques in an Orthogonal Projections to Latent Structures Discriminant Analysis. TGF-ß2 correlated positively to features of plaque stability and inversely to markers of plaque vulnerability. TGF-ß2 was the only isoform inversely correlated to the matrix-degrading matrix metalloproteinase-9 and inflammation in the plaque tissue. In vitro, TGF-ß2 pre-treatment reduced MCP-1 gene and protein levels as well as matrix metalloproteinase-9 gene levels and activity. Patients with plaques with high TGF-ß2 levels had a lower risk to suffer from future CV events. CONCLUSIONS: TGF-ß2 is the most abundant TGF-ß isoform in human plaques and may maintain plaque stability by decreasing inflammation and matrix degradation.

Increased proteolytic cleavage of osteoglycin is associated with a stable plaque phenotype and lower risk of cardiovascular events.

BACKGROUND AND AIMS: Extracellular matrix (ECM) remodeling is one of the key components in the formation of vulnerable atherosclerotic plaques and cardiovascular events. We recently showed that the full-length ECM-proteoglycan osteoglycin was associated with plaque vulnerability and future cardiovascular events. In the present study, we aimed to investigate the association of cleaved osteoglycin with plaque phenotype. METHODS: Two-hundred human carotid plaques were analyzed by immunohistochemistry. Cleaved osteoglycin and active caspase-3 were assessed by ELISA. ECM components (collagen, elastin and glycosaminoglycans) were assessed by colorimetric assays in plaque tissue homogenates. Matrix metalloproteinases (MMPs) were assessed using Milliplex. MMP-cleavage of osteoglycin and its effect on apoptosis were studied in vitro. Cardiovascular events were recorded during follow-up using national registries. RESULTS: Plaque levels of cleaved osteoglycin were significantly higher in asymptomatic plaques and correlated to α-actin plaque area, collagen, elastin and inversely to lipids, active. caspase-3 and a histological vulnerability index. Cleaved osteoglycin correlated to several MMPs, especially MMP-12, which was also shown to cleave osteoglycin in vitro. In vitro cleavage of osteoglycin was also associated with less smooth muscle cell apoptosis. Patients with high plaque levels of cleaved osteoglycin had a significantly lower risk to suffer from future cardiovascular events. CONCLUSIONS: The current study shows that cleaved osteoglycin is associated with a stable plaque phenotype and lower risk for future cardiovascular events. Potentially due to reduced cell apoptosis and ability to retain LDL. These results indicate that targeting the cleavage of osteoglycin may be a potential therapeutic strategy to stabilize plaques.

Plaque Vulnerability Index Predicts Cardiovascular Events: A Histological Study of an Endarterectomy Cohort.

Background The balance between stabilizing and destabilizing atherosclerotic plaque components is used in experimental studies and in imaging studies to identify rupture prone plaques. However, we lack the evidence that this balance predicts future cardiovascular events. Here we explore whether a calculated histological ratio, referred to as vulnerability index (VI), can predict patients at higher risk to suffer from future cardiovascular events. Methods and Results Carotid plaques and clinical information from 194 patients were studied. Tissue sections were used for histological analysis to calculate the VI (CD68 [cluster of differentiation 68], alpha-actin, Oil red O, Movat pentachrome, and glycophorin A). Postoperative cardiovascular events were identified through the Swedish National Inpatient Health Register (2005-2013). During the follow-up (60 months) 45 postoperative cardiovascular events were registered. Patients with a plaque VI in the fourth quartile compared with the first to third quartiles had significantly higher risk to suffer from a future cardiovascular event (P=0.0002). The VI was an independent predictor and none of the 5 histological variables analyzed separately predicted events. In the 13 patients who underwent bilateral carotid endarterectomy, the VI of the right plaque correlated with the VI of the left plaque and vice versa (r=0.7, P=0.01). Conclusions Our findings demonstrate that subjects with a high plaque VI have an increased risk of future cardiovascular events, independently of symptoms and other known cardiovascular risk factors . This strongly supports that techniques which image such plaques can facilitate risk stratification for subjects in need of more intense treatment.

Carotid Plaque Morphology is Similar in Patients with Reduced and Normal Renal Function.

BACKGROUND: Chronic Kidney Disease (CKD) is associated with an increased risk for cardiovascular events such as stroke. However, it is still unclear if decreased kidney function is associated with a vulnerable atherosclerotic plaque phenotype. To explore if renal function was associated with carotid plaque vulnerability we analyzed carotid plaques obtained at surgery from the Carotid Plaque Imaging Project (CPIP). METHODS: Patients were enrolled through the CPIP cohort. The indication for surgery was plaques with stenosis >70%, associated with ipsilateral symptoms or plaques with stenosis >80% not associated with symptoms. Transversal sections from the most stenotic plaque region were analyzed for connective tissue, calcium, lipids, macrophages, intraplaque hemorrhage, and smooth muscle cells. Homogenates were analyzed for collagen and elastin. RESULTS: Carotid endarterectomy specimens from 379 patients were obtained. The median GFR was 73 ml/min/1.73 m2. Plaque characteristics showed no significant association with eGFR, neither when eGFR was divided in CKD groups nor when eGFR was handled as a continuous variable and adjusting for other known risk factors (ie, age, diabetes, hypertension, and smoking). CONCLUSIONS: The higher risk of cardiovascular disease such as stroke in CKD is not associated with increased plaque vulnerability and other factors have to be sought.

The proteoglycan mimecan is associated with carotid plaque vulnerability and increased risk of future cardiovascular death.

BACKGROUND AND AIMS: A vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown. METHODS: 196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1ß were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-ß1, ß2 and ß3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up. RESULTS: Mimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death. CONCLUSIONS: This study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death.

Evidence for a protective role of placental growth factor in cardiovascular disease.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD.

Markers of Basement Membrane Remodeling Are Associated With Higher Mortality in Patients With Known Atherosclerosis.

Background Patients with atherosclerosis have a high risk of cardiovascular events and death. Atherosclerosis is characterized by accumulation of lipids, cells and extracellular matrix proteins in the intima. We hypothesized that dysregulated remodeling of the basement membrane proteins may be associated with clinical outcomes in patients with atherosclerosis. Methods and Results Neoepitope fragments of collagen type IV (C4M) and laminin ( LG 1M) were assessed by ELISA s in serum from 787 endarterectomy patients. Matrix metalloproteinase s were measured using proximity extension assay and correlated to C4M and LG 1M levels using Spearman correlations. A total of 473 patients were followed up for 6 years using national registers, medical charts, and telephone interviews. The incidence of cardiovascular events, cardiovascular mortality, and all-cause mortality were associated to levels of C4M and LG 1M using Kaplan-Meier curves and Cox regression analyses. A total of 101 patients had cardiovascular events, 39 died of cardiovascular mortality, and 64 patients died from all-cause mortality. C4M levels were increased in patients with symptomatic carotid atherosclerotic disease before surgery ( P=0.048). High C4M and LG 1M levels were associated with increased risk of all-cause mortality ( P=0.020 and 0.031, respectively) and predicted all-cause death together with glomerular filtration rate and diabetes mellitus. Conclusions High LG 1M and C4M levels were associated with all-cause mortality, together with glomerular filtration rate and diabetes mellitus. These novel biomarkers need further evaluation but might be tools to identify high-risk patients.

Expression of fibromodulin in carotid atherosclerotic plaques is associated with diabetes and cerebrovascular events.

AIMS: The small leucine-rich proteoglycans fibromodulin and lumican are functionally related extracellular matrix proteins involved in the regulation of collagen fiber formation. Fibromodulin-deficient apolipoprotein E-null mice have decreased vascular retention of lipids and reduced development of atherosclerosis suggesting that fibromodulin may influence the disease process. The aim of the present study was to investigate if fibromodulin and lumican are expressed in human carotid plaques and to determine if their expression is associated with the occurrence of preoperative symptoms and with risk for postoperative cardiovascular events. METHODS AND RESULTS: 153 plaques (51% symptomatic) obtained by carotid endarterectomy were included in this study. Plaque content was analyzed by immunohistochemistry and plaque cytokine content by multiplex technology. Fibromodulin and lumican were widely expressed in plaques and fibromodulin expression was significantly higher in symptomatic plaques. Expression of fibromodulin was significantly higher in plaques obtained from patients with diabetes and a high fibromodulin expression was associated with a higher incidence of post-operative cerebrovascular events, whereas no such associations were seen for lumican. Fibromodulin expression also correlated with plaque lipids and several pro-inflammatory cytokines. In addition, fibromodulin expression correlated with low levels of smooth muscle cells and the anti-inflammatory cytokine IL-10. CONCLUSIONS: These observations support previous experimental findings in mice for a role of fibromodulin in atherosclerosis and provide clinical evidence of the involvement of fibromodulin in the inflammatory processes that characterize atherosclerotic plaque vulnerability. They also suggest that this is of particular importance in diabetes.

Dystrophin deficiency reduces atherosclerotic plaque development in ApoE-null mice.

Dystrophin of the dystrophin-glycoprotein complex connects the actin cytoskeleton to basement membranes and loss of dystrophin results in Duchenne muscular dystrophy. We have previously shown injury-induced neointima formation of the carotid artery in mice with the mdx mutation (causing dystrophin deficiency) to be increased. To investigate the role of dystrophin in intimal recruitment of smooth muscle cells (SMCs) that maintains plaque stability in atherosclerosis we applied a shear stress-modifying cast around the carotid artery of apolipoprotein E (ApoE)-null mice with and without the mdx mutation. The cast induces formation of atherosclerotic plaques of inflammatory and SMC-rich/fibrous phenotypes in regions of low and oscillatory shear stress, respectively. Unexpectedly, presence of the mdx mutation markedly reduced the development of the inflammatory low shear stress plaques. Further characterization of the low shear stress plaques in ApoE-null mdx mice demonstrated reduced infiltration of CD3(+) T cells, less laminin and a higher SMC content. ApoE-null mdx mice were also found to have a reduced fraction of CD3(+) T cells in the spleen and lower levels of cytokines and monocytes in the circulation. The present study is the first to demonstrate a role for dystrophin in atherosclerosis and unexpectedly shows that this primarily involves immune cells.