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Publisher Copyright: © 2022 The Authors Background and aims: Glutamine synthetase (GLUL), the sole generator of glutamine, is a metabolic nexus molecule also involved in atherosclerosis. We recently demonstrated a 2.2-fold upregulation of GLUL mRNA in stroke-causing carotid plaques when compared with plaques from asymptomatic patients. Here we compared in the same cohort GLUL mRNA expression with plaque gross morphology, and the colocalization of immunodetectable GLUL protein with histopathological changes and molecular and mechanical mediators linked to plaque development. Methods: Endarterectomy specimens from 19 asymptomatic and 24 stroke patients were sectioned longitudinally and immunostained for GLUL, CD68, α-smooth muscle actin, iron, heme oxygenase-1 and CD163, and graded semiquantitatively in every 1 mm2. The amounts of cholesterol clefts and erythrocytes were graded. The fibrous cap thickness within each 1 mm2 area was measured. The association between the local pathological findings was analyzed by a hierarchical mixed modelling approach. Results: The previously found correlation between GLUL mRNA and clinical symptomatology was supported by the increased GLUL mRNA in diseased tissue and increased local GLUL immunoreactivity in areas with multiple different atherosclerotic changes. A longer symptom-to-operation time correlated with lower GLUL mRNA (Rs = −0.423, p=0.050) but few outliers had a significantly higher GLUL mRNA levels, which persisted throughout the post-symptomatic period. Plaque ulceration associated with 1.8-fold higher GLUL mRNA (p=0.006). Macrophages were the main GLUL immunoreactive cells. GLUL immunostaining colocalized with erythrocytes, iron, CD163, and heme oxygenase-1. The correlations between local variables were consistent in both asymptomatic and stroke-causing plaques. An inverse correlation was found between the fibrous cap thickness and local GLUL immunoreactivity (p=0.012). Considerable variability in interplaque expression pattern of GLUL was present. Conclusions: Our results link connect macrophage GLUL expression with carotid plaque features characterizing plaque vulnerability.

Mitochondrial reactive oxygen species (mROS) are central to physiology. While excess mROS production has been associated with several disease states, its precise sources, regulation, and mechanism of generationin vivoremain unknown, limiting translational efforts. Here we show that in obesity, hepatic ubiquinone (Q) synthesis is impaired, which raises the QH2/Q ratio, driving excessive mROS production via reverse electron transport (RET) from site IQin complex I. Using multiple complementary genetic and pharmacological modelsin vivowe demonstrated that RET is critical for metabolic health. In patients with steatosis, the hepatic Q biosynthetic program is also suppressed, and the QH2/Q ratio positively correlates with disease severity. Our data identify a highly selective mechanism for pathological mROS production in obesity, which can be targeted to protect metabolic homeostasis.

Fatty-acid binding protein 4 (FABP4) is implicated in the pathogenesis of cardiometabolic disorders. Pharmacological inhibition or genetic deletion of FABP4 improves cardiometabolic health and protects against atherosclerosis in preclinical models. As cardiovascular disease (CVD) is common in type 1 diabetes, we examined the role of FABP4 for the development of complications in type 1 diabetes, focusing on a functional, low-expression, variant (rs77878271) in the promoter of the FABP4 gene. For this, we assessed the risk of CVD, stroke, coronary artery disease (CAD), end-stage kidney disease (ESKD), and mortality using Cox proportional-hazard models for the FABP4 rs77878271 in 5,077 Finnish individuals with type 1 diabetes. The low-expression G-allele of rs77878271 increased the risk of CVD, independently of confounders. Findings were tested for replication in 852 Danish and 3,678 Finnish individuals with type 1 diabetes. In the meta-analysis, each G-allele increased the risk of stroke by 26% (p=0.04), CAD by 26% (p=0.006), and CVD by 17% (p=0.003). In Mendelian Randomization, a decrease in FABP4 increased CAD 2.4-fold. Hence, in contrast to the general population, the low-expression G-allele of rs77878271 increased CVD risk in type 1 diabetes, suggesting that genetically low FABP4 levels may be detrimental in the context of type 1 diabetes.

Fatty acid binding protein 4 (FABP4) is implicated in the pathogenesis of cardiometabolic disorders. Pharmacological inhibition or genetic deletion of FABP4 improves cardiometabolic health and protects against atherosclerosis in preclinical models. As cardiovascular disease (CVD) is common in type 1 diabetes, we examined the role of FABP4 in the development of complications in type 1 diabetes, focusing on a functional, low-expression variant (rs77878271) in the promoter of the

Liberation of energy stores from adipocytes is critical to support survival in times of energy deficit, however, uncontrolled or chronic lipolysis associated with insulin resistance and/or insulin insufficiency, disrupts metabolic homeostasis(1,2). Coupled to lipolysis is the release of a recently identified hormone, fatty acid-binding protein 4 (FABP4)(3). While circulating FABP4 levels have been strongly associated with cardiometabolic diseases in both preclinical models and humans(4–7), no mechanism of action has yet been described(8–10). Here, we show that hormonal FABP4 forms a novel functional hormone complex with Adenosine Kinase (ADK) and Nucleoside Diphosphate Kinase (NDPK) to regulate extracellular ATP and ADP levels. We identify a substantial impact of this hormone on beta-cells and given the central role of beta-cell function in both the control of lipolysis and development of diabetes, postulate that hormonal FABP4 is a key regulator of an adipose-beta-cell endocrine axis. Antibody-mediated targeting of this hormone complex improves metabolic outcomes, enhances beta-cell function, and preserves beta-cell integrity to prevent both type 1 and type 2 diabetes. Thus, the FABP4-ADK-NDPK complex, Fabtin, represents a previously unknown hormone and mechanism of action integrating energy status with the function of metabolic organs, representing a promising target against metabolic disease.

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 MessagesThis 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.

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.

The gelatinase enzymes, matrix metalloproteinases -2 and -9, are central mediators of blood–brain barrier disruption, actively studied in experimental models of neurological disease. Staining with in situ zymography (ISZ) allows visualization of gelatinase activity directly in brain tissue sections. However, quantifying microvascular gelatinase activity from ISZ-images is challenging and time consuming, as surrounding cell types often show significant confounding activity. We describe validation and performance of a workflow for automated image analysis of cerebromicrovascular gelatinase activity, now released for open-access use. In comparison to manual analysis, the automated workflow showed superior accuracy, was faster to execute and allows for more detailed analysis of heterogeneity in the microvasculature. We further suggest recommendations for quantifying and reporting this type of activity in experimental studies, focusing on ischemic stroke.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated Lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular Lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular Lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density Lipoprotein, very-Low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been Lipolytically modified in vitro induced intracellular Lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular Lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

Objective: Intraplaque hemorrhages (IPH) may predispose to unstable atherosclerotic disease and its atherothrombotic complications, ischemic stroke and coronary syndromes. However, the discriminative value of IPH has been limited in histological and imaging studies suggesting that confounding factors modulate the response to IPH. We studied whether common variants of haptoglobin (Hp), which facilitates the removal of free hemoglobin and protects tissues from heme-iron induced oxidative damage, would modify the inflammatory response to IPH and the risk of unstable carotid stenosis (CS) and major cardiovascular diseases. Methods: We genotyped Hp polymorphism in 91 patients with a high-grade CS from Helsinki Carotid Endarterectomy Study (HeCES) and in 1417 individuals from Health 2000, a Finnish epidemiological cross-sectional health survey, and determined heme oxygenase-1 (HO1) expression in relation to Hp genotypes in carotid plaques. Results: In the Health 2000 cohort, Hp genotype frequencies were 0.143 (hp1-1), 0.486 (hp1-2) and 0.371 (hp2-2) consistent with HardyeWeinberg equilibrium and those reported from other Caucasian populations. Among patients with unstable CS, the frequency of hp2-2 genotype was higher than in the control population (0.516 vs. 0.371, P ¼ 0.025). Hp genotypes correlated with HO1 expression in the plaque (r ¼ 0.47, P ¼ 0.027). In the Health 2000 cohort, hp2 allele was associated with an increased risk of major cardiovascular diseases (ischemic stroke, TIA, myocardial infarction, coronary heart disease) with an adjusted OR of 1.46 (95% CI 1.03e2.06). Conclusion: Common haptoglobin variants modulate the inflammatory response to IPH and associate with the risk of unstable carotid stenosis and major ischemic cardiovascular events.

Introduction: Haptoglobin (Hb) is an acute phase plasma protein which binds free hemoglobin and protects tissues from heme-iron induced oxidative damage. Hp gene exists in two variant alleles, Hp1 and Hp2 giving rise to three protein isoforms, hp1-1, hp1-2 and hp2-2, which differ in their efficiency to limit oxidative stress. Previously, Hp variants have been associated with the risk of ischemic stroke subtypes, Hp1 variant with lacunar stroke and Hp2 with large-artery atherosclerotic stroke. Hypothesis: Our aim was to scrutinize previous associations to stroke subtypes and, further, to test association of Hp variants with the extent of white matter lesions (WML) and incidence of cardiovascular death after first-ever ischemic stroke. Methods: Hp was genotyped by PCR and gel electrophoresis in Helsinki Stroke Aging Memory Study (SAM, n=378 with DNA) and Cardiovascular diseases -subcohort of Health 2000 Survey (n=1426), who served as population controls. Results: Hp genotype frequencies were 15.6% (Hp1-1), 44.2% (Hp1-2) and 40.2% (Hp2-2), within Hardy-Weinberg equilibrium and equal to those described in other Caucasian populations. There were no significant differences in Hp genotypes between stroke subtypes classified according to TOAST within SAM or compared to population controls. Hp alleles did not associate with the extent of WML. In follow-up, the carriers of Hp2 allele (Hp1-2 and Hp2-2) had substantially higher rate of cardiac deaths (24.5% vs 8.5%, P=0.006) and a trend towards more fatal strokes (23.5% vs 13.6%, P=0.122). The combined risk of ischemic cardiovascular death was 2.4-fold increased in Hp2 carriers (95%CI 1.28-4.43) even after adjustment for major cardiovascular risk factors (age, gender, hypertension, diabetes, atrial fibrillation, smoking, high LDL cholesterol). Conclusions: Hp2 allele is associated with increased risk of ischemic cardiovascular death after first-ever ischemic stroke. This may result from the impaired capacity of Hp2-containing protein isoforms to alleviate oxidative stress in the vasculopathic arterial tree.

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. Electronic supplementary material The online version of this article (doi:10.1007/s00109-011-0773-z) contains supplementary material, which is available to authorized users.

Paracoccus denitrificans is a soil bacterium which can respire aerobically and also denitrify if oxygen is absent. Both processes are highly dependent on copper enzymes and copper is therefore likely to be an essential trace element for the bacterium. If copper is not easily available, a copper-acquisition mechanism would be highly beneficial. In this paper, we have addressed the question of whether Paracoccus secretes a copper-acquisition compound functionally analogous to that found in some methanotrophs. Bacteria were grown both in copper-containing and copper-deficient denitrification media, cells were removed by centrifugation and the supernatant was analysed using chromatography and spectroscopy. Bacterial growth yield in the absence of copper was 70-80% of that in the copper-containing medium. A notable difference between the two culture conditions was that spent copper-deficient medium was pigmented, whereas the copper-containing medium was not. Spectrophotometry indicated that a red compound with an absorption maximum at 405 nm was produced under copper-limited conditions. In addition to the strong 405 nm maximum, the visible spectrum of the purified red molecule had weaker maxima at 535 nm and 570 nm, features typical of metallated tetrapyrroles. Mass spectrometry showed that the purified pigment had a molecular mass of 716.18. Moreover, the fine structure of the mass spectrum suggested the presence of zinc and was consistent with the chemical formula of C(36)H(36)N(4)O(8)Zn. The presence of zinc was also demonstrated using inductively coupled plasma atomic emission spectroscopy. Fragmentation analysis with mass spectrometry showed the release of consecutive 59 Da fragments, assignable to four -CH(2)-COOH moieties. Thin layer chromatography as well as NMR analysis of the C-13/N-15 labelled red pigment suggested that it is predominantly zinc coproporphyrin III with a minor fraction of metal-free coproporphyrin III. We propose that in a copper-poor environment P. denitrificans secretes coproporphyrin III for copper chelation and subsequent uptake of the bound copper into the cell. Consistent with this idea, cell yields of copper-deficient cultures grown in the presence of 1 microM copper-coproporphyrin III were 90-95% of the yields of cultures grown in the normal copper-containing media. Coproporphyrin III may work as a copper-acquisition compound in P. denitrificans.

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.

Cholesterol is a critical nutrient requiring tight constraint in the endoplasmic reticulum (ER) due to its uniquely challenging biophysical properties. While the mechanisms by which the ER defends against cholesterol insufficiency are well described, it remains unclear how the ER senses and effectively defends against cholesterol excess. Here, we identify the ER-bound transcription factor nuclear factor erythroid 2 related factor-1, Nrf1/Nfe2L1, as a critical mediator of this process. We show that Nrf1 directly binds to and specifically senses cholesterol in the ER through a defined domain and that cholesterol regulates Nrf1 turnover, processing, localization, and activity. In Nrf1 deficiency, in vivo cholesterol challenges induce massive hepatic cholesterol accumulation and damage, which is rescued by replacing Nrf1 exogenously. This Nrf1-mediated mechanism involves the suppression of CD36-driven inflammatory signaling and derepression of liver X receptor activity. These findings reveal Nrf1 as a guardian of cholesterol homeostasis and a core component of adaptive responses to excess cellular cholesterol.

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 P P 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.

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.

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.

Objectives: Haptoglobin (Hp) is a plasma protein which binds free hemoglobin (Hb) protecting tissues from iron-induced oxidative damage and promoting Hb clearance via macrophage CD163 receptor. There are two common alleles for Hp (Hp 1 and Hp 2), which influence it’s capacity to bind Hb and induce downstream signaling via CD163+ macrophages. We have previously shown that the CD163-heme oxygenase-1 (HO1) pathway is upregulated in symptomatic carotid stenosis (CS) and other groups have associated the Hp 2-2 genotype with cardiovascular complications in diabetics. Here, our objective was to study whether Hp polymorphism influences the risk of symptomatic CS and major cardiovascular events in the general population. Methods: We genotyped Hp polymorphism in 90 patients with a high-grade CS (≥ 70%) from Helsinki Carotid Endarterectomy Study (HeCES) and in 1426 individuals with carotid ultrasound examination data from the Health 2000 Survey, an epidemiological cross-sectional health survey carried out in Finland in the year 2000. Results: In the Health 2000 population, the Hp genotype frequencies were 0.170 (Hp 1-1), 0.445 (Hp 1-2) and 0.386 (Hp 2-2) consistent with Hardy-Weinberg equilibrium and with those reported from caucasian populations. In the CS patients, the genotype frequencies were skewed towards the Hp 2-2 genotype but not significantly. However, among the patients with symptomatic CS (ipsilateral TIA or stroke), the frequency of Hp 2-2 genotype was significantly higher than in the control population (0.528 vs 0.386, P =0.044). Hp genotypes correlated with HO1 protein expression in the plaque (r=0.46, P = 0.038) but not with iron. In the Health 2000 population, Hp 2 allele was associated with an increased risk of major cardiovascular events (major coronary heart disease event, ischemic stroke or TIA; 13.3% vs 8.3, P =0.032) and the increased risk was present regardless of diabetes. Hp 2 allele was not associated with carotid IMT or presence of plaques. Conclusions: Hp 2 allele associates with the risk of symptomatic CS and major ischemic cardiovascular events. This is likely due to the defective ability of Hp 2 to block oxidative reactions mediated by iron/heme, to clear free Hb present in intraplaque hemorrhages as well as the effects of the Hb-Hp 2 complex on intracellular signaling cascades via the CD163 receptor.

Background and Purpose— Perivascularly positioned cerebral mast cells (MC) have been shown to participate in acute blood-brain barrier disruption and expansive brain edema following experimental transient cerebral ischemia. However, the underlying molecular mechanisms remain unknown. Because proteolytic gelatinase enzymes, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to have a central role in compromising the integrity of the blood-brain barrier following ischemia, we examined whether cerebral MCs influence gelatinase activity in ischemic cerebral microvasculature. Methods— Rats underwent 60 minutes of middle cerebral artery occlusion followed by 3-hour reperfusion, and were treated with a MC-stabilizing (cromoglycate), or MC-degranulating (compound 48/80) agent, or vehicle. Genetically manipulated, MC-deficient WsRc Ws/Ws rats and their wild-type littermates (WT) underwent the same procedures. Cerebral edema and extravasation of Evans blue albumin were measured. Gelatinase activity was visualized by in situ zymography and was quantified with computerized high-throughput image and data analysis. Results— Activated MCs showed secretion of gelatinase-positive granules. Genetic MC deficiency decreased global gelatinase-active area (−69%, compared with WT; P P =0.002). MC stabilization with cromoglycate decreased the percentage of microvessels with high gelatinase activity (−36% compared with saline; P P r =0.84; P r =0.61; P =0.02). Conclusions— Our data demonstrate that cerebral MCs participate in regulation of acute microvascular gelatinase activation and consequent blood-brain barrier disruption following transient cerebral ischemia.

Apoptotic cell death contributes to neuronal loss in the penumbral region of brain infarction. Activated caspase-3 (ACA-3) cleaves proteins including poly(ADP-ribose) polymerase-1 (PARP-1) important in DNA repair, thus promoting apoptosis. Overactivation of PARP-1 depletes NAD+ and ATP, resulting in necrosis. These cell death phenomena have been investigated mostly in experimental animals. We studied an autopsy cohort of 13 fatal ischemic stroke cases (symptoms 15 h to 18 days) and 2 controls by immunohistochemical techniques. The number of PARP-1 immunoreactive neurons was highest in the periinfarct area. Nuclear PARP-1 correlated with increasing neuronal necrosis (P = 0.013). Cytoplasmic PARP-1 correlated with TUNEL in periinfarct and core areas (P = 0.01). Cytoplasmic cleaved PARP-1 was inversely correlated with increasing necrotic damage (P = 0.001). PAR-polymers were detected in neurons confirming enzymatic activity of PARP-1. Cytoplasmic ACA-3 correlated with death receptor Fas (r s = 0.48; P = 0.005). In conclusion, the confirmation of the same pathways of cell death than previously described in experimental animal models encourages neuroprotective treatments acting on these mediators also in human stroke.

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.