Carotid atherosclerotic plaques from symptomatic stroke patients share the molecular fingerprints to develop in a neoplastic fashion: A microarray analysis study - ScienceDirect

Neuroscience

Volume 131, Issue 2, 2005, Pages 359-374

Neuroscience

https://doi.org/10.1016/j.neuroscience.2004.08.058 Get rights and content

Abstract

Identification of genetic mechanisms that promote the onset of stroke and transient cerebral ischemic attack symptoms in carotid atherosclerotic patients would further our understanding of the pathophysiology of this disease and could lead to new pharmacological and molecular therapies. Using Affymetrix Human Genome 230 GeneChip set, the present study evaluated the gene expression differences in geometrically similar carotid artery plaque samples extricated from six symptomatic stroke patients and four asymptomatic patients. There was no significant difference in the degree of stenosis between the two groups. Of the 44,860 transcripts analyzed, 289 (approximately 0.6% of the total transcripts) were differentially expressed between the plaques from the symptomatic and asymptomatic groups (236 were expressed more abundantly and 53 were expressed less abundantly in the symptomatic group). Of the 236 transcripts expressed more abundantly in the symptomatic plaques, 71% (167 transcripts) indicate an active cell proliferation and neoplastic process. These include oncogenes, growth factors, tumor promoters, tumor markers, angiogenesis promoters, transcription factors, RNA splicing factors, RNA processing proteins, signal transduction mediators and those that control the metabolism. Real-time polymerase chain reaction confirmed the increased expression of 63 transcripts in the symptomatic plaques. The other groups of transcripts expressed more abundantly in the symptomatic plaques are those that control ionic homeostasis, those that participate in the progression of degenerative neurological diseases (Alzheimer’s disease, amyotrophic lateral sclerosis and Huntington’s disease) and epilepsy. This indicates that symptomatic plaques are molecularly and biochemically more active than the asymptomatic plaques, or active plaque growth precipitates stroke symptoms.

Section snippets

Plaque samples

Patients scheduled for endarterectomy were enrolled to this plaque study after describing the study and obtaining the informed consent. The patients underwent preoperative magnetic resonance imaging that identified the plaque location and the degree of stenosis. Of the 10 plaques used in this study, six are from symptomatic stroke patients and four were from asymptomatic patients. There was no significant difference in the age, body mass index, degree of stenosis, previous heart attacks,

Results

There was no statistically significant difference in the total number of transcripts expressed between the plaques from the symptomatic and the asymptomatic groups. Of the 44,860 transcripts represented on the Affymetrix Human Genome 230 GeneChip set (22,215 on chip A and 22,645 transcripts on chip B), 19,342±2956 transcripts obtained a present call in various plaque samples. Of these, 289 (approximately 0.6% of the total transcripts analyzed) were differentially expressed between the

Discussion

Although carotid atherosclerotic plaques are commonly seen in adult humans, only some of them induce stroke symptomology. Higher rates of smooth muscle cell proliferation, connective tissue formation, cholesterol deposition, calcification, bacterial infection and extravasation of inflammatory cells are known to promote a faster plaque growth (Ismail et al., 1999; Scott, 2002; Leskinen et al., 2003), but the molecular mechanisms that make a plaque become symptomatic leading to rupture and

Acknowledgments

These studies were funded by grants from the National Institutes of Health, American Heart Association, University of Wisconsin Medical School and the University of Wisconsin Department of Neurological Surgery to R.V. and R.J.D. K. Bowen and V. Dhodda helped with the sample preparation and the staff of the UW Gene Expression Center conducted the hybridizations.

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Cerebrovascular disease (CVD) is the second most common cause of cognitive impairment and dementia in aged population. CVD presents in a myriad number of clinical ways based on the functional location of pathology. While primary clinical emphasis has been placed on motor, speech and visual deficits, vascular cognitive decline is a vastly under recognized and devastating condition afflicting millions of Americans. CVD, a disease of the blood vessels that supply blood to brain involves an integration between small and large vessels. Cerebral large vessel diseases (LVD) are associated with atherosclerosis, artery-to-artery embolism, intracardiac embolism and a large vessel stroke leading to substantial functional disability. Cerebral small vessel disease (SVD) is critically involved in stroke, brain hemorrhages, cognitive decline and functional loss in elderly patients. An evolving understanding of cellular and molecular mechanisms emphasizes that inflammatory vascular changes contribute to systemic pathologic conditions of the central nervous systems (CNS), with specific clinical presentations including, cognitive decline. Advances in an understanding of pathophysiology of disease processes and therapeutic interventions may help improve outcomes. This review will focus on large and small vessels diseases and their relationship to vascular cognitive decline, atherosclerosis, stroke, and inflammatory neurodegeneration. We will also emphasize the molecular and cellular mechanisms, as well as genetic and epigenetic factors associated with LVD and SVD.
Gene and Protein Expression of Chemokine (C-C-Motif) Ligand 19 is Upregulated in Unstable Carotid Atherosclerotic Plaques
2016, European Journal of Vascular and Endovascular Surgery
Citation Excerpt :
The amount of specimen and the quality of RNA needed for qRT-PCR validation is also less than what is required for microarray, making it a potentially more accurate and accessible platform when carried forward for clinical applications. Gene expression studies in carotid atherosclerosis using microarray have been used previously to identify differential expression between (1) symptomatic versus asymptomatic patient groups;20–22 (2) postautopsy versus live tissue;23 (3) cap versus intima;24 (4) plaque versus normal arterial wall;25 and (5) intraplaque stable and unstable regions.26 However, limitations of some of these studies have included small numbers used for both array work and for validation, and that, in some studies, pathway-specific arrays were examined, and therefore a whole transcriptomic approach was not used.
The aim was to investigate the expression of genes associated with carotid plaque instability and their protein products at a local and systemic level.
Carotid plaques from 24 patients undergoing carotid endarterectomy (CEA) were classified as stable or unstable using clinical, histological, ultrasound, and transcranial Doppler criteria, and compared using whole genome microarray chips. Initial results of differentially expressed genes were validated by quantitative reverse transcriptase polymerase chain reaction in an independent group of 96 patients undergoing CEA. The protein product of genes significantly differentially expressed between patients with stable and unstable plaques were analysed by plaque immunohistochemistry and serum protein quantification by enzyme-linked immunosorbent assay on a further independent cohort.
Expression of chemokine (c-c-motif) ligand 19 (CCL19) was significantly upregulated in plaques from patients with clinically unstable disease (p < .001). Cathepsin G expression was upregulated in histologically unstable plaques (p = .04). Serum concentration of CCL19 was significantly higher in patients with clinically unstable plaques (p = .02). Immunohistochemical staining for CCL19 demonstrated positive staining in histologically and clinically unstable plaques (p = .03). CCL19 also co-localised with CD3⁺ T-cell lymphocytes in the core region, around where CCL19 was expressed.
CCL19 is significantly overexpressed in patients with unstable carotid atherosclerotic plaques and may be a possible novel biomarker for identifying high-risk patients in whom more urgent intervention may be indicated.
Upregulation of ANGPTL4 messenger RNA and protein in severely calcified carotid plaques
2014, Journal of Stroke and Cerebrovascular Diseases
In carotid atherosclerotic lesions, calcified plaques are thought to be stable and to evoke very few symptoms. However, the molecular activity in calcified plaques and their clinical significance have not been fully clarified yet.
Carotid plaques from 18 endarterectomy patients were classified into high- and low-calcified plaques on the basis of Agatston calcium score. Twelve plaques were investigated for the alteration of gene expression by microarray analysis and real-time polymerase chain reaction (PCR) and 6 other plaques underwent protein assessment to elucidate the difference in molecular biological activity between the groups.
Microarray analysis demonstrated 93 angiogenesis or growth factor–related transcripts that are reliably expressed (175 probe sets). Among them, angiopoietin-like protein 4 (ANGPTL4) expression was significantly elevated, whereas fibroblast growth factor receptor 2 (FGFR2) expression was significantly suppressed. Quantitative messenger RNA analysis was performed with real-time PCR. Augmented or decreased protein expression of each gene was confirmed by Western blotting analysis and immunohistochemistry.
In high-calcified plaques, ANGPTL4 might be upregulated for antiangiogenic modulating function together with the downregulation of FGFR2, contributing to the stability of the plaques.
Histologically unstable asymptomatic carotid plaques have altered expression of genes involved in chemokine signalling leading to localised plaque inflammation and rupture
2013, European Journal of Vascular and Endovascular Surgery
Citation Excerpt :
Papaspyridonos et al.24 looked at histologically ‘stable’ vs ‘unstable’ plaques but samples included patients who were both symptomatic and asymptomatic. Previous microarray studies comparing carotid atherosclerotic plaques in symptomatic and histologically ‘unstable’ plaques have alluded to the role of angiogenesis,23 including hypoxia driven angiogenesis,25 inflammation,18,19 cytokine and chemokine signalling,26 apoptosis,27,28 alterations in lipid metabolism,21,22 osteogenesis,28,29 proteolysis and collagenolysis,29 and changes in iron metabolism in plaque progression.20 This study further adds evidence regarding the role of altered expression of genes related to inflammation, proteolysis and chemokine signalling, but also suggests an additional role for disturbances in transcription, spliceosome pathway and cellular components related to the endoplasmic reticulum.
Many studies have evaluated histological and gene expression profiles in TIA/stroke patients after onset of symptoms, but there is limited understanding as to how these plaque related features interact before symptom onset. In particular, no studies have evaluated differential gene expression in histologically unstable (vs stable plaques) in neurologically asymptomatic patients.
Nine asymptomatic patients had their plaques scored blindly by two independent Histopathologists using the AHA plaque scoring system. RNA extracted from the plaques was hybridised onto a whole genome microarray. Analysis was performed using GenomeStudio (v1.0) and the DAVID bioinformatics resource (v6.7).
Three plaques were histologically unstable (Grade 2/3), while six were stable (Grade 0/1). 346 differentially expressed genes (>1.3 fold, P < 0.05) were identified (293 down-regulated and 53 up-regulated) between stable and unstable plaques. Genes related to chemokine and protein signalling (pro-inflammatory/pro-apoptotic) were identified to have high enrichment scores (>1.3) and were significantly up-regulated in unstable (asymptomatic) plaques.
The findings confirm the intuitively held belief that changes in chemokine and protein signalling may be associated with acute plaque disruption and precede the onset of symptoms. Once validated, these genes could therefore become targets for innovative medical treatments in the future or could help identify asymptomatic patients with histologically unstable plaques that would benefit from surgical intervention.
All's well that transcribes well: Non-coding RNAs and post-stroke brain damage
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The mammalian genome is replete with various classes of non-coding (nc) RNA genes. Many of them actively transcribe, and their relevance to CNS diseases is just beginning to be understood. CNS is one of the organs in the body that shows very high ncRNAs activity. Recent studies demonstrated that cerebral ischemia rapidly changes the expression profiles of different classes of ncRNAs: including microRNA, long noncoding RNA and piwi-interacting RNA. Several studies further showed that post-ischemic neuronal death and/or plasticity/regeneration can be altered by modulating specific microRNAs. These studies are of interest for therapeutic development as they may contribute to identifying new ncRNA targets that can be modulated to prevent secondary brain damage after stroke.
Increased expression of fatty acid binding protein 4 and leptin in resident macrophages characterises atherosclerotic plaque rupture
2013, Atherosclerosis
Citation Excerpt :
While this approach should yield greater specificity for the pathophysiological process of plaque rupture, the results could still be to a degree confounded by the differences in cell composition between stable and ruptured lesions. Indeed, genes that would be correlated with the quantity of macrophages present in a lesion (for example inflammatory mediators, matrix metalloproteinases, pro-apoptotic factors) have been consistently reported as differentially expressed in such studies of whole stable and ruptured plaques [4–10]. For example, Puig et al. identified a gene expression signature of inflamed whole plaques which closely overlapped the expression profile of laser microdissected macrophages from a single plaque [10].
Resident macrophages play an important role in atheromatous plaque rupture. The macrophage gene expression signature associated with plaque rupture is incompletely defined due to the complex cellular heterogeneity in the plaque. We aimed to characterise differential gene expression in resident plaque macrophages from ruptured and stable human atheromatous lesions.
We performed genome-wide expression analyses of isolated macrophage-rich regions of stable and ruptured human atherosclerotic plaques. Plaques present in carotid endarterectomy specimens were designated as stable or ruptured using clinical, radiological and histopathological criteria. Macrophage-rich regions were excised from 5 ruptured and 6 stable plaques by laser micro-dissection. Transcriptional profiling was performed using Affymetrix microarrays. The profiles were characteristic of activated macrophages. At a false discovery rate of 10%, 914 genes were differentially expressed between stable and ruptured plaques. The findings were confirmed in fourteen further stable and ruptured samples for a subset of eleven genes with the highest expression differences (p < 0.05). Pathway analysis revealed that components of the PPAR/Adipocytokine signaling pathway were the most significantly upregulated in ruptured compared to stable plaques (p = 5.4 × 10⁻⁷). Two key components of the pathway, fatty-acid binding-protein 4 (FABP4) and leptin, showed nine-fold (p = 0.0086) and five-fold (p = 0.0012) greater expression respectively in macrophages from ruptured plaques.
We found differences in gene expression signatures between macrophages isolated from stable and ruptured human atheromatous plaques. Our findings indicate the involvement of FABP4 and leptin in the progression of atherosclerosis and plaque rupture, and suggest that down-regulation of PPAR/adipocytokine signaling within plaques may have therapeutic potential.

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