Petra Ijäs

Microarray Analysis Reveals Overexpression of CD163 and HO-1 in Symptomatic Carotid Plaques

Objective—We studied by microarray analysis whether symptomatic and asymptomatic carotid plaques from the same patient differ in gene expression and whether the same changes are present in an independent sample set. Methods and Results—Carotid plaques from four patients with bilateral high-grade stenosis, one being symptomatic and the other asymptomatic, were analyzed on Affymetrix U95Av2 arrays. 33 genes showed >1.5-fold change between symptomatic and asymptomatic plaques in an intraindividual comparison with FDR ranging from 0.28 to 0.40. Three genes involved in iron-heme homeostasis, CD163, HO-1, and transferrin receptor, were further analyzed in 40 independent plaques. HO-1 (fold-change 1.93, 95%CI 1.04 to 3.94, P=0.040) and CD163 (1.58, 1.11 to 2.40, P=0.013) mRNAs were again induced, and also HO-1 protein was overexpressed in symptomatic plaques (4.38, 1.54 to 12.20, P=0.024). The expression of HO-1 and CD163 correlated with tissue iron content but iron itself was not associated with the symptom status. Conclusions—Symptomatic plaques show overexpression of CD163 and HO-1 both in intraindividual and interindividual comparison. Their expression correlates with iron deposits but asymptomatic and symptomatic plaques from isolated patients do not differ in macroscopic hemorrhages or iron deposits. We suggest that symptomatic plaques show a more pronounced induction of CD163 and HO-1 in response to plaque hemorrhages.

Adipophilin Expression Is Increased in Symptomatic Carotid Atherosclerosis: Correlation With Red Blood Cells and Cholesterol Crystals

Background and Purpose— Adipophilin is an adipose differentiation–related protein expressed in lipid-containing cells. Using DNA microarray analysis, we previously found the adipophilin gene (ADFP) to be overexpressed in symptomatic carotid plaques (CP). This led us to further examine the role of adipophilin in carotid atherosclerosis relative to symptom status. Methods— Ninety-eight high-grade (>70%) CPs were obtained in carotid endarterectomy. The relative expression of ADFP mRNA was measured by quantitative real-time RT-PCR, and the relative amount of adipophilin protein was quantified with Western blotting. Detailed topographical correlations with extravasated red blood cells and extracellular cholesterol crystals were obtained by means of immunohistochemistry. Results— The relative expression of ADFP mRNA was increased in symptomatic compared with asymptomatic CPs at both the mRNA level (1.82±0.19[SE] versus 1.25±0.15, P=0.012) and the protein level (1.04±0.23 versus 0.46±0.14, P=0.043). Adipophilin colocalized with macrophage foam cells, extravasated red blood cells (P<0.0001), and cholesterol crystals (P<0.0001), and its expression associated with macroscopic ulceration of CP (P<0.0001). Conclusions— Intraplaque hemorrhages may contribute to intracellular lipid accumulation and consequent adipophilin expression. Because adipophilin blocks cholesterol efflux from lipid-laden cells, they may die and develop a necrotic lipid core, thereby destabilizing the plaque.

Gene expression differences between stroke-associated and asymptomatic carotid plaques

Atherosclerotic carotid stenosis is an important risk factor for stroke. Carotid plaques (CPs) causing stroke may present a distinct type of molecular pathology compared with transient ischemic attack (TIA)-associated or asymptomatic plaques. We compared the gene expression profiles of CPs from stroke patients (n = 12) and asymptomatic patients (n = 9), both with similar risk factors and severity of carotid stenosis (>70%). Sixty probes showed over 1.5-fold expression difference at 5% false discovery rate. Functional clustering showed enrichment of genes in 51 GO categories and seven pathways, the most significant of which relate to extracellular-matrix interaction, PPAR gamma signaling, scavanger receptor activity, and lysosomal activity. Differential expression of ten genes was confirmed in an extended replication group (n = 43), where the most significant expression differences were found in CD36 (2.1-fold change, p = 0.005), CD163 (1.7-fold change, p = 0.007) and FABP4 (2.2-fold change, p = 0.015). These include four genes not previously linked to plaque destabilization: GLUL (2.2-fold change, p = 0.016), FUCA1 (2.2-fold change, p = 0.025), IL1RN (1.6-fold change, p = 0.034), and S100A8 (2.5-fold change, p = 0.047). Strong correlations were found to plaque ulceration, plaque hemorrhage, and markers of apoptosis and proliferation (activated caspase 3, TUNEL, and Ki67). Protein expression of these genes was confirmed by immunohistochemistry and was found in the atheromatous areas of CPs critical for plaque destabilization. This study presents a comprehensive transcriptional analysis of stroke-associated CPs and demonstrates a significant transcriptome difference between stroke-associated and asymptomatic CPs. Follow-up studies on the identified genes are needed to define whether they could be used as biomarkers of symptomatic CPs or have a role in plaque destabilization.

Low-expression variant of fatty acid-binding protein 4 favors reduced manifestations of atherosclerotic disease and increased plaque stability.

Background—Fatty acid–binding protein 4 (FABP4 or aP2 in mice) has been identified as a key regulator of core aspects of cardiometabolic disorders, including lipotoxic endoplasmic reticulum stress in macrophages. A functional promoter polymorphism (rs77878271) of human FABP4 gene has been described resulting in reduced FABP4 transcription. Methods and Results—We investigated the effects of this low-expression variant of FABP4 on cardiovascular morbidity and carotid atherosclerosis on a population level (n=7491) and in patient cohorts representing endarterectomized patients with advanced carotid atherosclerosis (n=92) and myocardial infarction (n=3432). We found that the low-expression variant was associated with decreased total cholesterol levels (P=0.006) with the largest reduction in variant allele homozygotes. Obese variant allele carriers also showed reduced carotid intima-media thickness (P=0.010) and lower prevalence of carotid plaques (P=0.060). Consistently, the variant allele homozygotes showed 8-fold lower odds for myocardial infarction (P=0.019; odds ratio, 0.12; 95% confidence interval, 0.003–0.801). Within the carotid plaques, the variant allele was associated with a 3.8-fold reduction in FABP4 transcription (P=0.049) and 2.7-fold reduction in apoptosis (activated caspase 3; P=0.043). Furthermore, the variant allele was enriched to patients with asymptomatic carotid stenosis (P=0.038). High FABP4 expression in the carotid plaques was associated with lipid accumulation, intraplaque hemorrhages, plaque ulcerations, and phosphoactivated endoplasmic reticulum stress markers. Conclusions—Our results reveal FABP4 rs77878271 as a novel variant affecting serum total cholesterol levels and cardiovascular risk. A therapeutic regimen reducing FABP4 expression within the atherosclerotic plaque may promote lesion stability through modulation of endoplasmic reticulum stress signaling, and attenuation of apoptosis, lipid burden, and inflammation.

Editor's Choice - Safety of Carotid Endarterectomy After Intravenous Thrombolysis for Acute Ischaemic Stroke: A Case-Controlled Multicentre Registry Study

OBJECTIVE Few studies have been published on the safety of carotid endarterectomy (CEA) after intravenous thrombolysis (IVT). Registry reports have been recommended in order to gather large study groups. DESIGN A retrospective, registry based, case controlled study on prospectively gathered data from Sweden, the capital region of Finland, and from Denmark, including 30 days of follow up. METHODS The study group was a consecutive series of 5526 patients who had CEA for symptomatic carotid artery stenosis during a 4.5 year period. Among these, 202 (4%) had IVT prior to surgery, including 117 having CEA within 14 days, and 59 within 7 days of thrombolysis. IVT as well as CEA were performed following established guidelines. The median time from index symptom to CEA was 12 days (range 0-130, IQR 7-21). RESULTS The 30 day combined stroke and death rate was 3.5% (95% CI 1.69-6.99) for those having IVT + CEA, 4.1% (95% CI 3.46-4.39) for those having CEA without previous IVT (odds ratio 0.84 [95% CI 0.39-1.81]), 3.4% (95% CI 1.33-8.39) for those having IVT + CEA within 14 days, and 5.1% (95% CI 1.74-13.91) for those having IVT + CEA within 7 days. CONCLUSION Data on the time from symptoms to CEA in patients not having IVT, Rankin score, degree of stenosis, and cerebral imaging were not available. Despite its weaknesses, this study reasserts that CEA can be performed within the recommended 2 weeks of the onset of symptoms and IVT without increasing the risk of peri-operative stroke or death. Centres and vascular registries are recommended to continue monitoring changes in patient characteristics, lead times, and major complications after CEA in general, with a special focus on those who have undergone a prior thrombolysis.

An Imbalance Between CD36 and ABCA1 Protein Expression Favors Lipid Accumulation in Stroke-Prone Ulcerated Carotid Plaques

Background— CD36 is a macrophage scavenger receptor mediating the uptake of modified lipoproteins, whereas the ABCA1 transporter counteracts this effect by mediating cellular lipid efflux. Based on a DNA microarray, we previously found that the CD36 and ABCA1 genes were overexpressed in symptom-causing carotid plaques (CP) compared with nonsymptom-causing CP. To evaluate their role in CP destabilization, we conducted detailed immunohistochemical studies on the localization of lipids, CD36 and ABCA1 proteins, extravasated red blood cells, and atheromatous/necrotic tissue. Methods— Ninety-two high-grade (>70%) stenosing CP obtained from carotid endarterectomy were Oil-red-O–stained for evaluation of neutral lipids. Subgroups of nonsymptom-causing and symptom-causing CP (n=42) were further analyzed by immunostaining adjacent histological sections against CD36 and ABCA1 and examining them microscopically. Results— When compared with nonsymptom-causing CP, the amount of extracellular lipid and the expression of CD36 protein were elevated in symptom-causing CP, but no difference was found in ABCA1 expression. These observations were also confirmed when ulcerated and nonulcerated CP were compared. In ulcerated CP, CD36 protein expression was higher than that of ABCA1, and the opposite was true in nonulcerated CP. CD36 colocalized with extravasated red blood cells and atheromatous or necrotic areas in the various types of CP. Conclusions— Our results suggest that an imbalance between lipid influx (CD36) and efflux (ABCA1) favors lipid accumulation in macrophages of ulcerated CP, thus contributing to plaque destabilization. Furthermore, colocalization of CD36 protein with red blood cells suggests that intraplaque hemorrhages may contribute to the lipid load and thus the stability of CP.

Endothelial Apoptosis Does Not Determine Symptom Status in Carotid Artery Disease

Background: We examined the hypothesis that endothelial denudation in advanced carotid plaques (CPs) occurs by increased apoptosis of endothelial cells (ECs) using scanning electron microscopy (SEM) as well as markers of cellular proliferation and apoptosis in advanced symptomatic CPs (SCPs) and asymptomatic CPs (ACPs). Methods: 93 consecutive patients underwent carotid endarterectomy. Five additional specimens were studied by SEM. We performed TUNEL assays, and immunostaining against Fas receptor (FasR), Fas ligand (FasL), activated caspase 3 (ACA3) and Ki-67. Results: SEM revealed morphological changes consistent with EC detachment. Surprisingly, ACA3 positivity was more pronounced on the endothelium of ACPs (4.6 ± 0.7% of total EC count) than on SCPs (3.3 ± 0.7%, p = 0.049), and was found to correlate positively with nuclear Ki-67 expression (rs = 0.275, p = 0.040). FasL expression was significantly increased on the endothelium of SCPs compared with ACPs (66.4 ± 4.4 vs. 53.9 ± 4.5%, p = 0.047). Conclusions: Absence of increased positivity of apoptotic markers dismisses apoptosis as a dominant mechanism underlying endothelial detachment of SCPs. Rather, increased ACA3 with co-expression of Ki-67 in ACPs might suggest that renewal of endothelium by active cell turnover may contribute to clinically silent evolution of plaques with preserved EC integrity. These observations may assist in designing novel therapies to prevent endothelial decay and symptom generation in advanced carotid artery disease.

Life-threatening coronary disease is prevalent in patients with stenosing carotid artery disease

Background Atherosclerosis affects several vascular trees systemically and though surgical plaque removal diminishes the risk of stroke in patients with carotid stenosis, they still face a risk of other atherothrombotic complications like myocardial infarction and premature death. Aims and/or hypothesis This study was designed to reveal the long-term risk of death and atherothrombotic events following carotid endarterectomy. Methods Eighty-nine previously (1997–2000) endarterectomized carotid patients (56–92 years) were followed up to 15·2 years. Causes of death, cardiovascular events (stroke, transient ischemic attack, acute myocardial infarction), comorbidities, and medications were recorded and analyzed by Cox regression analysis. Four population controls and four controls with coronary disease (n = 712) were selected for each case from a population cohort for age- and gender-matched analysis. Results At the end of follow-up, 41 (44·6%) patients had died and 48 were alive. Ten patients (24,4%) died due to acute myocardial infarction and one (2,4%) due to stroke. Nineteen (21%) patients had an acute myocardial infarction, 12 (13%) had a stroke, 13 (15%) had a transient ischemic attack, and 5 (6%) had other atherothrombotic events. The risk of death was 5·7-fold in diabetics (P < 0·001) and 3·9-fold in smokers (P < 0·001). Patients who did not use statins had 5·0-fold, and irregular users 3·3-fold risk of death compared with active users (P = 0·005 and P = 0·001, respectively). The major factors associated with acute myocardial infarction were diabetes (6·0-fold risk, P = 0·004), bilateral carotid disease (3·5-fold risk, P = 0·014), and lack of statin use (4·4-fold risk, P = 0·038). Compared with population controls, carotid patients had a 4·4-fold risk of acute myocardial infarction (P = 0·002). Conclusions Endarterectomized carotid patients have a high risk of acute myocardial infarction and death, and need an intensified cardiovascular disease-risk-lowering treatment. Although asymptomatic, the evaluation of prognostically significant myocardial ischemia should be considered in these high-risk patients. Eventually, a clinical trial is needed to address whether carotid patients would benefit from early intervention.

Haptoglobin Hp2 Variant Promotes Premature Cardiovascular Death in Stroke Survivors

Background and Purpose— Haptoglobin (Hp) is an acute phase plasma protein protecting tissues from oxidative damage. It exists in 2 variant alleles (hp1/hp2) giving rise to 3 protein isoforms with different biochemical properties and efficiency to limit oxidative stress. We previously found that hp2 variant is associated with stroke risk in the patients with carotid stenosis and the risk of ischemic cardiovascular events in a general population cohort. This study examined the hypothesis that Hp genotype is associated with general cardiovascular risk in patients with stroke. Methods— Hp was genotyped in SAM study (Helsinki Stroke Aging Memory, n=378). A total of 1426 individuals ascertained from a nationally representative cross-sectional health survey served as population controls. Results— Hp genotype frequencies were 15.6% (hp1-1), 44.2% (hp1-2), and 40.2% (hp2-2) in patients with stroke. During a mean of 7.5-year follow-up after first-ever stroke, hp2 carriers had a substantially higher rate of cardiac deaths (24.5% versus 8.5%; P=0.006) and a trend toward more fatal strokes (23.5% versus 13.6%; P=0.122). The combined risk of ischemic cardiovascular deaths was 2.4-fold higher among hp2 carriers (95% confidence interval, 1.28–4.43) after adjustment for major cardiovascular risk factors. Conclusions— Hp2 allele is associated with premature ischemic cardiovascular deaths after first-ever ischemic stroke.

Morphology and histology of silent and symptom-causing atherosclerotic carotid plaques – Rationale and design of the Helsinki Carotid Endarterectomy Study 2 (the HeCES2)

Abstract Introduction: Every fifth ischemic stroke is caused by thromboembolism originating from an atherosclerotic carotid artery plaque. While prevention is the most cost-effective stroke therapy, antiplatelet and cholesterol-lowering drugs have a ceiling effect in their efficacy. Therefore, discovery of novel pathophysiologic targets are needed to improve the primary and secondary prevention of stroke. This article provides a detailed study design and protocol of HeCES2, an observational prospective cohort study with the objective to investigate the pathophysiology of carotid atherosclerosis. Materials and Methods: Recruitment and carotid endarterectomies of the study patients with carotid atherosclerosis were performed from October 2012 to September 2015. After brain and carotid artery imaging, endarterectomised carotid plaques (CPs) and blood samples were collected from 500 patients for detailed biochemical and molecular analyses. Findings to date: We developed a morphological grading for macroscopic characteristics within CPs. The dominant macroscopic CP characteristics were: smoothness 62%, ulceration 61%, intraplaque hemorrhage 60%, atheromatous gruel 59%, luminal coral-type calcification 34%, abundant (44%) and moderate (39%) intramural calcification, and symptom-causing “hot spot” area 53%. Future plans: By combining clinically oriented and basic biomedical research, this large-scale study attempts to untangle the pathophysiological perplexities of human carotid atherosclerosis. Key Messages This article is a rationale and design of the HeCES2 study that is an observational prospective cohort study with the objective to investigate the pathophysiology of carotid atherosclerosis. The HeCES2 study strives to develop diagnostic algorithms including radiologic imaging to identify carotid atherosclerosis patients who warrant surgical treatment. In addition, the study aims at finding out new tools for clinical risk stratification as well as novel molecular targets for drug development.