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Cardiovascular diseases due to atherosclerosis are the leading cause of death globally. We aimed to investigate the potentially altered gene and pathway expression in advanced peripheral atherosclerotic plaques in comparison to healthy control arteries. Gene expression analysis was performed (Illumina HumanHT-12 version 3 Expression BeadChip) for 68 advanced atherosclerotic plaques (15 aortic, 29 carotid and 24 femoral plaques) and 28 controls (left internal thoracic artery (LITA)) from Tampere Vascular Study. Dysregulation of individual genes was compared to healthy controls and between plaques from different arterial beds and Ingenuity pathway analysis was conducted on genes with a fold change (FC) > ±1.5 and false discovery rate (FDR) < 0.05. 787 genes were significantly differentially expressed in atherosclerotic plaques. The most up-regulated genes were osteopontin and multiple MMPs, and the most down-regulated were cell death-inducing DFFA-like effector C and A (CIDEC, CIDEA) and apolipoprotein D (FC > 20). 156 pathways were differentially expressed in atherosclerotic plaques, mostly inflammation-related, especially related with leukocyte trafficking and signaling. In artery specific plaque analysis 50.4% of canonical pathways and 41.2% GO terms differentially expressed were in common for all three arterial beds. Our results confirm the inflammatory nature of advanced atherosclerosis and show novel pathway differences between different arterial beds., Competing Interests: The authors declare no competing financial interests.

Background: Kindlins (FERMT) are cytoplasmic proteins required for integrin (ITG) activation, leukocyte transmigration, platelet aggregation and thrombosis. Characterization of kindlins and their association with atherosclerotic plaques in human(s) is lacking., Methods and Results: Exploratory microarray (MA) was first performed followed by selective quantitative validation of robustly expressed genes with qRT-PCR low-density array (LDA). In LDA, ITGA1 (1.30-fold, p = 0.041) and ITGB3 (1.37-fold, p = 0.036) were upregulated in whole blood samples of patients with coronary artery disease (CAD) compared to healthy controls. In arterial plaques, both robustly expressed transcript variants of FERMT3 (MA: 5.90- and 3.4-fold; LDA: 3.99-fold, p < 0.0001 for all) and ITGB2 (MA: 4.81- and 4.92-fold; LDA: 5.29-fold, p < 0.0001 for all) were upregulated while FERMT2 was downregulated (MA: -1.61-fold; LDA: -2.88-fold, p < 0.0001 for both). The other integrins (ITGA1, ITGAV, ITGB3, ITGB5) were downregulated. All these results were replicated in at least one arterial bed. The latter FERMT3 transcript variant associated with unstable plaques (p = 0.0004). FERMT3 correlated with M2 macrophage markers and in hierarchical cluster analysis clustered with inflammatory and macrophage markers, while FERMT2 correlated with SMC-rich plaque markers and clustered with SMC markers. In confocal immunofluorescence analysis, FERMT3 protein colocalized with abundant CD68-positive cells of monocytic origin in the atherosclerotic plaques, while co-localization of FERMT3 with HHF35 indicative of smooth muscle cells was low., Conclusions: Kindlin-3 (FERMT3) is upregulated in atherosclerotic, especially unstable plaques, mainly in cells of monocytic origin and of M2 type. Simultaneous upregulation of ITGB2 suggests a synergistic effect on leukocyte adherence and transmigration into the vessel wall., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Macrophage (MΦ) infiltration and smooth muscle cell (SMC) proliferation are hallmarks of atherosclerosis and unstable plaques. Neuroimmune guidance cue 1 (netrin-1 [NTN1]) plays a critical role controlling MΦ trafficking and SMC activation. Characterization of expression of NTN1 and its receptors and their association with plaque stability in human(s) is lacking., Methods and Results: The expression of NTN1 and its receptors did not differ in either whole blood or circulating monocytes from patients with coronary artery disease (n=55) compared with healthy controls (n=45). However, NTN1 was downregulated (-2.9-fold; P<0.0001) and UNC5B upregulated (2.2-fold; P<0.0001) in atherosclerotic plaques (n=68), whereas there were no differences in other NTN1 receptors compared with histologically normal controls (n=28). Increased UNC5B expression is associated with histologically more stable plaques (P=0.011). NTN1 expression correlated positively with SMC markers and signatures and negatively with inflammatory markers and M1 and especially M2 signatures in the atherosclerotic plaques. UNC5B clustering correlated positively with inflammatory and MΦ markers. NTN1 protein colocalized with CD68-positive cells of monocytic origin and muscle-actin-specific-antibody (HHF3)-positive cells indicative of SMCs in the plaques and only with SMCs in the control samples. NTN1 protein was highly expressed in the intimal layer of the control vessels., Conclusions: Present findings provide support for the hypothesis that dysregulation of expression of NTN1 in SMCs and its chemorepulsive receptor UNC5B in macrophages are involved in the development of atherosclerosis and unstable plaques.

Aims: This study was set up to compare the accuracy, sensitivity and specificity of gene expression results between the Illumina HumanHT-12 v3 Expression BeadChip (GWE) and the TaqMan qRT-PCR low-density array (LDA) in atherosclerotic plaques., Methods: Gene expression levels of 196 genes were determined from 22 atherosclerotic samples and 6 controls with both the GWE and the LDA., Results: The accuracies of GWE in comparison to that of qRT-PCR for absolute fold changes (FC) 1.2, 1.6, 2.0 and 3.0 between cases and controls were 73.5%, 79.1%, 72.4% and 60.7%, respectively. The correlation of expression measurements between these methods was good (r = 0.87, y = 0.151 + 0.586x), and the Bland-Altman plot showed that for highly up- or down-regulated transcripts, GWE yields lower absolute FC values than LDA., Conclusion: Gene expression results obtained with the GWE are replicated with high accuracy in LDA, even for technically demanding atherosclerotic samples., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: Atherosclerosis is a complex disease with hundreds of genes influencing its progression. In addition, the phenotype of the disease varies significantly depending on the arterial bed., Methodology/principal Findings: We characterized the genes generally involved in human advanced atherosclerotic (AHA type V-VI) plaques in carotid and femoral arteries as well as aortas from 24 subjects of Tampere Vascular study and compared the results to non-atherosclerotic internal thoracic arteries (n=6) using genome-wide expression array and QRT-PCR. In addition we determined genes that were typical for each arterial plaque studied. To gain a comprehensive insight into the pathologic processes in the plaques we also analyzed pathways and gene sets dysregulated in this disease using gene set enrichment analysis (GSEA). According to the selection criteria used (>3.0 fold change and p-value <0.05), 235 genes were up-regulated and 68 genes down-regulated in the carotid plaques, 242 genes up-regulated and 116 down-regulated in the femoral plaques and 256 genes up-regulated and 49 genes down-regulated in the aortic plaques. Nine genes were found to be specifically induced predominantly in aortic plaques, e.g., lactoferrin, and three genes in femoral plaques, e.g., chondroadherin, whereas no gene was found to be specific for carotid plaques. In pathway analysis, a total of 28 pathways or gene sets were found to be significantly dysregulated in atherosclerotic plaques (false discovery rate [FDR] <0.25)., Conclusions: This study describes comprehensively the gene expression changes that generally prevail in human atherosclerotic plaques. In addition, site specific genes induced only in femoral or aortic plaques were found, reflecting that atherosclerotic process has unique features in different vascular beds.

Background: Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave proproteins into mature end products. Previously, MBTPS1 and PCSK9 have been shown to regulate cholesterol metabolism and LDL receptor recycling, whereas FURIN and PCSK5 have been suggested to inactivate lipases and regulate inflammation in atherosclerosis. Here, we systematically analyzed the expression of PCSKs and their targets in advanced atherosclerotic plaques., Methods and Results: Microarray and quantitative real-time PCR experiments showed that FURIN (42.86 median fold, p = 2.1e-8), but no other PCSK, is universally overexpressed in the plaques of different vascular regions. The mRNA expression screen of PCSK target proteins in plaques identified many known factors, but it also identified the significant upregulation of the previously overlooked furin-processed B cell activating cytokines APRIL (TNFSF13, 2.52 median fold, p = 3.0e-5) and BAFF (TNFSF13B, 2.97 median fold, p = 7.6e-6). The dysregulation of FURIN did not associate with its htSNPs or the previously reported regulatory SNP (-229, rs4932178) in the promoter. Immunohistochemistry experiments showed the upregulation of FURIN in the plaque lymphocytes and macrophages where it was co-expressed with BAFF/TNFSF13B and APRIL/TNFSF13., Conclusions: Our data unequivocally show that FURIN is the primary PCSK that is dysregulated in the immune cells of advanced human atherosclerotic plaques, which implies a role for this enzyme in plaque pathology. Therefore, drugs that inhibit FURIN in arteries may modulate the course of this disease., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: MicroRNAs are small non-coding RNAs that inversely regulate their target gene expression. The whole miRNA profile of human atherosclerotic plaques has not been studied previously. The aim of this study was to investigate the miRNA expression profile in human atherosclerotic plaques as compared to non-atherosclerotic left internal thoracic arteries (LITA), and to connect this expression to the processes in atherosclerosis., Methods: The miRNA expression profiles of six LITAs and 12 atherosclerotic plaques obtained from aortic, carotid, and femoral atherosclerotic arteries from Tampere Vascular Study were analyzed. The analyses were performed with Agilent's miRNA Microarray. The expression levels of over 4-fold up-regulated miRNAs were verified with qRT-PCR from a larger population (n=50). Messenger RNA levels were analyzed with Illumina's Expression BeadChip to study miRNA target expression., Results: Ten miRNAs were found to be differently expressed in atherosclerotic plaques when compared to controls (p<0.05). The expression of miR-21, -34a, -146a, -146b-5p, and -210 was verified and found to be significantly up-regulated in atherosclerotic arteries versus LITAs (p<0.001, fold changes 4.61, 2.55, 2.87, 2.82, and 3.92, respectively). Several predicted targets of these miRNAs were down-regulated, and gene set enrichment analysis showed several pathways which could be differently expressed due to this miRNA profile., Conclusions: The microRNA expression profile differs significantly between atherosclerotic plaques and control arteries. The most up-regulated miRNAs are involved in processes known to be connected to atherosclerosis. Interfering with the miRNA expression in the artery wall is a potential way to affect atherosclerotic plaque and cardiovascular disease development., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)