Glutamine synthetase in human carotid plaque macrophages associates with features of plaque vulnerability : An immunohistological study

Publisher Copyright: © 2022 The AuthorsBackground 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.Peer reviewe

The Low-Expression Variant of FABP4 Is Associated With Cardiovascular Disease in 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 FABP4 gene. For this, we assessed the risk of CVD, stroke, coronary artery disease (CAD), end-stage kidney disease, and mortality using Cox proportional hazards 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, independent 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 1-SD unit decrease in FABP4 increased risk of CAD 2.4-fold. Hence, in contrast with the general population, among patients with type 1 diabetes the low-expression G allele of rs77878271 increased CVD risk, suggesting that genetically low FABP4 levels may be detrimental in the context of type 1 diabetes.Peer reviewe

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

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.Peer reviewe

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties

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.Peer reviewe

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

Stroke-Associated Carotid Atherosclerosis : A Role for FABP4 in Atheroma Vulnerability and Cardiometabolic Risk

Carotid artery disease is a common cause of stroke, with approximately one in five ischemic strokes in the carotid artery distribution stemming from thromboembolisms caused by an unstable atherosclerotic carotid plaque (CP). Despite the fact that carotid atherosclerosis can lead to cerebrovascular complications, some patients afflicted by a high-grade carotid stenosis (>70%) remain asymptomatic. This divergence in clinical courses captures one of the most pressing questions in cardiovascular medicine: what are the key molecules and pathways decisive to local atheroma vulnerability and connected with disabling clinical outcomes? In the first paper, we utilized a genome-wide approach to screen for gene expression changes in stroke-associated CPs to identify genes and pathways related to atheroma vulnerability and symptom generation. This approach was successful in identifying several significant expression changes in novel genes and in genes already connected with atherosclerosis development, suggesting that the local atheroma-derived gene expression patterns are closely connected to the patient s clinical phenotype. A detailed data-driven approach, using three independent data pre-processing methods, identified fatty acid-binding protein 4 (FABP4) as the lead hit overexpressed in stroke-associated CPs, thus linking FABP4 and its transcriptional activity to atheroma vulnerability. Immunohistochemical analysis revealed the most prominent expression of FABP4 protein in the histological regions of atheroma vulnerability, coinciding with the abundance of lipid-laden macrophages and thus connecting the overexpression of FABP4 with lipid accumulation and inflammation within the CPs. In the second paper, we focused on the molecular mechanisms through which increased FABP4 expression could regulate atheroma vulnerability. As causality is difficult to infer from associative expression data we adapted a genetic approach searching for variants that could regulate the expression of FABP4. Using a naturally occurring low-expression variant (rs77878271) we were able to link genetically reduced atheroma expression of FABP4 to endoplasmic reticulum (ER) stress signaling, and the attenuation of apoptosis, with high expression contributing to increased lipid burden and inflammation. We discovered that the low-expression variant of FABP4 was associated with lower circulating total cholesterol levels, with the lowest levels in obese (body mass index, BMI ≥ 30) minor allele homozygotes. Furthermore, the variant allele carriers showed obesity-related reduction in subclinical markers of atherosclerosis, manifested as reduced carotid intima-media thickness (IMT) and lower prevalence of CPs. Most importantly, we found that the local atheroma effects of FABP4 penetrated to end point level, as the minor allele homozygotes showed 8-fold lower odds for myocardial infarction (MI) and enrichment of the variant in patients with clinically asymptomatic carotid artery disease. Obesity-related reduction of total cholesterol levels observed in the low-expression variant carriers pointed to a metabolic component mediated by genetically reduced FABP4 expression. Based on the discoveries made in FABP4-deficient mouse models, we hypothesized that genetically reduced FABP4 expression could lead to sustained enhancement of adipocentric de novo lipogenesis (DNL), which could in part explain the obesity-related phenotypes also in man. We found that the low-expression variant of FABP4 was associated with circulating markers of enhanced lipolysis and reduced circulating FABP4 protein levels. The variant allele carriers showed a propensity for higher BMI, with evidence for enhancement of DNL, improved beta-cell function, and reduced obesity-related type 2 diabetes (T2D) risk. These results imply that FABP4-regulated production of adipocyte-derived lipid signals during DNL in the low-expression variant carriers may in part lead to enhanced beta-cell function and reduced obesity-related T2D risk. In this thesis work, we identified FABP4 as a key molecule at the crossroads of metabolic and inflammatory responses, potentially involved in local atheroma vulnerability and connected with cardiometabolic outcomes. Most likely the role of FABP4 in atherosclerosis is multifaceted, converging with the passage of time within the artery atheroma, and culminating in the regulation of inflammatory responses affecting cell survival, acute plaque vulnerability and ultimately cardiovascular risk. These data implicate FABP4 as a potential pharmacological target for prevention of cardiometabolic complications connected with atherosclerosis and obesity.Sydän- ja verisuonitaudit ovat maailman yleisin kuolinsyy. Vuonna 2012 tämän sairausryhmän nimiin kirjattiin 17,5 miljoonaa kuolemantapausta, mikä tarkoittaa kolmea kuolemaa kymmenestä. Näiden kuolemien kavala taustavaikuttaja on perimän ohjastama ja raskaan ruokakulttuurin sekä nuokkuvan länsimaisen elämäntavan hiljalleen edistämä valtimokovettumatauti eli ateroskleroosi. Tyypillisin riskitekijä tälle tulehdukselliselle valtimosairaudelle on veren kohonnut LDL-kolesterolipitoisuus. Sopivissa riskitekijäolosuhteissa kolesteroli kertyy valtimoseinämän sisäkerrokseen ja muodostaa ennen pitkää veren virtausta häiritsevän ahtauman, joka voi oireillessaan revetä ja aiheuttaa sydän- tai aivoinfarktin. Aivoihin verta vievissä kaulavaltimoissa ateroskleroottiset ahtaumat muodostuvat tyypillisesti sisemmän kaulavaltimon haaraumakohtaan. Arviolta joka viidennen aivoinfarktin taustalla onkin kaulavaltimotauti: kaulavaltimoon muodostunut ahtauma on repeytyessään johtanut aivoinfarktin syntyyn. Kaulavaltimotauti ei kuitenkaan aina oireile – toisilla ahtaumat pysyvät oireettomina läpi elämän, kun taas toisilla ne voivat halvaannuttaa. Tässä väitöstutkimuksessa suurennuslasin alla oli itse aivoinfarktin aiheuttanut kaulavaltimoahtauma ja polttopisteessä ne vielä toistaiseksi tuntemattomat syyt, jotka johtivat ahtauman oireisuuteen. Kartoitimme geenien aktiivisuuseroja aivoinfarktin aiheuttaneiden ja oireettomina säilyneiden kaulavaltimoahtaumien välillä. Genomin kattavassa vertailussa noin 20 000 geenin joukosta avainasemaan siivilöityi rasvahappojen sitojaproteiini 4 -geeni (engl. fatty acid-binding protein 4, FABP4), jonka aktiivisuus oli lisääntynyt aivoinfarktin aiheuttaneissa ahtaumissa. Valtimoahtaumissa ylivilkkaasti toimiva FABP4-geeni liittyi oireelliseen valtimokovettumatautiin, mutta syy-seuraussuhteen selvittäminen vaatisi geenin hiljentämisen ihmisessä. Tunnistimmekin potilasaineistossamme geenimuunnoksen, joka johti perinnöllisesti matalampaan FABP4-geenin aktiivisuuteen, myös kaulavaltimoahtaumissa. Kysymys oli siis FABP4-geenin vähemmän aktiivisesta, ”hiljaisesta” variantista. Yllättäen joka kymmenes suomalainen osoittautui tämän geenimuodon kantajaksi. Löytö mahdollisti luonnon esivalmisteleman kokeen sen selvittämiseksi, suojaako hiljaisen geenimuunnoksen välittämä perinnöllisesti matalampi FABP4-geenin aktiivisuus valtimokovettumataudin aiheuttamilta sydän- ja aivoinfarkteilta. Tulokset osoittivat, että hiljaisen FABP4-muunnoksen molemmilta vanhemmiltaan perineillä henkilöillä sydäninfarktit olivat lähes 8 kertaa muuta väestöä harvinaisempia. Vastaavasti hiljaista muunnosta kantavat henkilöt, joilla oli merkittävä kaulavaltimoahtauma, säästyivät muita useammin aivoinfarkteilta. Väestötasolla havaitsimme, että lihavilla FABP4-muunnoksen kantajilla alkavasta valtimokovettumataudista viestivät merkit – kuten kaulavaltimoseinämän paksuuntuminen ja matala-asteiset valtimoahtaumat – olivat muuta väestöä harvinaisempia. Hiljaisen FABP4-muunnoksen ja veren matalamman kokonaiskolesterolipitoisuuden välillä osoittautui olevan yhteys, ja tämä yhteys korostui eniten lihavilla henkilöillä jotka olivat perineet geenimuunnoksen sekä isältä että äidiltään. Tämä uusi löydös valtimokovettumataudin merkittävimmän riskitekijän ja perinnöllisesti madaltuneen FABP4-geenin aktiivisuuden välillä on yhtenevä hiljaisen FABP4-muunnoksen kantajilla havaitun sydän- ja aivotapahtumien vähentymisen kanssa. Kaulavaltimoahtaumien tarkempi tutkiminen paljasti, että FABP4-geeni oli aktiivisin kolesterolia ahmineissa tulehdussoluissa. FABP4 liittyikin kaulavaltimoahtaumissa korkeampaan kolesterolimäärään, ohjelmoidusta solukuolemasta kertovien viestinproteiinien aktiivisuuteen sekä taudin oireellisuudesta kertoviin ahtaumien sisäisiin verenvuotoihin ja sen pinnan haavaumiin. Ahtaumien kolesterolia ahmineiden tulehdussolujen rasvaperäinen solustressi kytkeytyi FABP4-geenin aktiivisuuteen ja vastaavasti hiljaisen FABP4-muunnoksen kantajilla tämän solustressin lopputulema eli ohjelmoitunut solukuolema oli kolme kertaa vähäisempää kuin verrokeilla. Rasvakudoksessa FABP4-proteiinin rooli on merkittävä, sillä se on vastuussa rasvasolun sisäisestä rasvahappokuljetuksesta. Kiinnostavaa kyllä, FABP4-poistogeeniset hiiret ovat suojassa valtimokovettumataudin lisäksi myös diabeteksen kehittymiseltä siitäkin huolimatta että ne ovat verrokkisisaruksiaan lihavampia. Voisiko FABP4-geenin hiljainen muunnos suojata kantajiaan myös diabetekselta? Kuten FABP4-poistogeenisyydellä hiiressä, havaitsimme että ihmisen hiljaisella FABP4-muunnoksella oli yhteys lihavuuteen, mutta näillä henkilöillä sairastuminen lihavuuteen kytkeytyvään tyypin 2 diabetekseen oli vähäisempää. Tässä väitöskirjatyössä tunnistimme FABP4-geenin yliaktiivisuuden yhteyden oireilevan kaulavaltimoahtauman ja aivoinfarktin välillä. Vastaavasti havaitsimme, että FABP4-geenin hiljainen muunnos, joka hillitsee geenin ilmentymistä, liittyi vakaampaan ahtaumatyyppiin ja pienentyneeseen sydän- ja aivoinfarktien esiintyvyyteen. Geenimuunnos oli yhteydessä lihavuuteen, mutta liittyi lihavilla henkilöillä matalampiin kolesterolitasoihin ja suojasi lihavuuteen liittyvän tyypin 2 diabeteksen puhkeamiselta. Tulosten perusteella FABP4 voi olla mahdollinen uuden lääkeaineen kehittelyn kohde sydän- ja aivoinfarktien sekä diabeteksen ennaltaehkäisyyn erityisesti lihavilla henkilöillä

Warfarin Treatment Is Associated to Increased Internal Carotid Artery Calcification

Background: Long-term treatment with the vitamin K antagonist warfarin is widely used for the prevention of venous thrombosis and thromboembolism. However, vitamin K antagonists may promote arterial calcification, a phenomenon that has been previously studied in coronary and peripheral arteries, but not in extracranial carotid arteries. In this observational cohort study, we investigated whether warfarin treatment is associated with calcification of atherosclerotic carotid arteries. Methods: Overall, 500 consecutive patients underwent carotid endarterectomy, 82 of whom had received long-term warfarin therapy. The extent of calcification was assessed with preoperative computed tomography angiography, and both macroscopic morphological grading and microscopic histological examination of each excised carotid plaque were performed after carotid endarterectomy. Results: Compared with non-users, warfarin users had significantly more computed tomography angiography-detectable vascular calcification in the common carotid arteries (odds ratio 2.64, 95% confidence interval 1.51-4.63, P < 0.001) and even more calcification in the internal carotid arteries near the bifurcation (odds ratio 18.27, 95% confidence interval 2.53-2323, P < 0.001). Histological analysis revealed that the intramural calcified area in plaques from warfarin users was significantly larger than in plaques from non-users (95% confidence interval 3.36-13.56, P = 0.0018). Conclusions: Long-lasting warfarin anticoagulation associated with increased calcification of carotid atherosclerotic plaques, particularly in locations known to be the predilection sites of stroke-causing plaques. The clinical significance of this novel finding warrants further investigations.Peer reviewe