Molecular mediators linking stroke and carotid artery disease

Carotid artery disease is the most prevalent etiologic precursor of ischemic stroke, which is a major health hazard and the second most common cause of death in the world. If a patient presents with a symptomatic high-grade (>70%) stenosis in the internal carotid artery, the treatment of choice is carotid endarterectomy. However, the natural course of radiologically equivalent carotid lesions may be clinically quite diverse, and the reason for that is unknown. It would be of utmost importance to develop molecular markers that predict the symptomatic phenotype of an atherosclerotic carotid plaque (CP) and help to differentiate vulnerable lesions from stable ones. The aim of this study was to investigate the morphologic and molecular factors that associate with stroke-prone CPs. In addition to immunohistochemistry, DNA microarrays were utilized to identify molecular markers that would differentiate between symptomatic and asymptomatic CPs. Endothelial adhesion molecule expression (ICAM-1, VCAM-1, P-selectin, and E-selectin) did not differ between symptomatic and asymptomatic patients. Denudation of endothelial cells was associated with symptom-generating carotid lesions, but in studies on the mechanism of decay of endothelial cells, markers of apoptosis (TUNEL, activated caspase 3) were found to be decreased in the endothelium of symptomatic lesions. Furthermore, markers of endothelial apoptosis were directly associated with those of cell proliferation (Ki-67) in all plaques. FasL expression was significantly increased on the endothelium of symptomatic CPs. DNA microarray analysis revealed prominent induction of specific genes in symptomatic CPs, including those subserving iron and heme metabolism, namely HO-1, and hemoglobin scavenger receptor CD163. HO-1 and CD163 proteins were also increased in symptomatic CPs and associated with intraplaque iron deposits, which, however, did not correlate with symptom status itself. ADRP, the gene for adipophilin, was also overexpressed in symptomatic CPs. Adipophilin expression was markedly increased in ulcerated CPs and colocalized with extravasated red blood cells and cholesterol crystals. Taken together, the phenotypic characteristics and the numerous possible molecular mediators of the destabilization of carotid plaques provide potential platforms for future research. The denudation of the endothelial lining observed in symptomatic CPs may lead to direct thromboembolism and maintain harmful oxidative and inflammatory processes, predispose to plaque microhemorrhages, and contribute to lipid accumulation into the plaque, thereby making it vulnerable to rupture.Valtimonkovetustaudin aiheuttama ahtauma kaulavaltimossa, on yksi tärkeimmistä aivoverenkiertohäiriön aiheuttajista. Aivojen paikallisesta hapenpuutteesta johtuva halvausoire, aivoverenkiertohäiriö (AH), on toiseksi yleisin kuolinsyy maailmassa. Paras ennaltaehkäisevä hoito on kirurginen ahtauman poistaminen kaulavaltimosta, jos potilaalla todetaan vahva-asteinen (>70%) oireinen ahtauma kaulavaltimossaan. Röntgentutkimuksissa havaitut samanasteiset kaulavaltimoahtaumat (KVA) eivät kuitenkaan käyttädy samalla tavoin; toiset aiheuttavat AH:tä ja toiset eivät, ja syytä tälle ei tiedetä. Olisi tärkeää pystyä kehittämään molekulaarisia markkereita, jotka voisivat ennakoida KVA:n mahdollista oireilua, ja näinollen pystyä erottamaan rauhalliset ahtaumat niistä, jotka todennäköisesti aiheuttavat AH:n. Tämän tutkimuksen tarkoitus oli tutkia niitä ahtauman rakenteellisia ja molekulaarisia tekijöitä, jotka liittyvät oireiden ilmenemiseen. Sen lisäksi, että pyrimme tunnistamaan näitä tekijöitä immunohistokemiallisilla menetelmillä, käytimme myös DNA mikrosiruja, pystyäksemme löytämään eroja oireisten ja oireettomien KVA:n välillä. Adheesiomolekyylien (ICAM-1, VCAM-1, P-selektiini ja E-selektiini) ilmeneminen oli samanlaista sekä oireisissa että oireettomissa ahtaumissa. Havaitsimme, että oireisissa KVA:issa oli enemmän endoteelisolukerroksen irtoamista. Tutkittaessa endoteelisolujen ohjelmoitua solukuolemaa (apoptoosia), havaitsimme sitä olevan vähemmän oireisissa KVA:issa. Lisäksi apoptoosimarkkerien ilmenemisellä oli suora yhteys solun lisääntymisestä kertoviin markkereihin. Fas ligandin (yksi tärkeimmistä solun apoptoosia laukaisevista tekijöistä) ilmeneminen oli lisääntynyt oireisten KVA:n endoteelilla. DNA mikrosiruanalyysit paljastivat oireisissa KVA:issa merkittävän geeni-induktion mm. raudan ja hemin aineenvaihduntaan liittyvien geenien (HO-1 ja CD163) kohdalla. Myös HO-1 ja CD163 geenien proteiinituotteet olivat lisääntyneet oireisissa KVA:ssa, ja assosioituivat vapaan raudan määrään. ADRP, adipofiliinin geeni, oli yli-ilmentynyt oireisissa KVA:issa. Adipofiliinin ilmeneminen oli suurempaa oireissa KVA:issa, ja adipofiliinia oli havaittavissa samoilla alueilla kolesterolikiteiden ja suonen seinämään purkautuneiden punasolujen kanssa. Tämä tutkimus paljasti, että AH:tä aiheuttavien ja hiljaisten, oireita aiheuttamattomien KVA:ien välillä on eroja mm. ahtauman pinnan endoteelisolukerroksen eheydessä, ja raudan ja hemin aineenvaihduntaan, sekä rasva-aineenvaihduntaan liittyvissä tekijöissä. Endoteelin puuttuminen voi johtaa suoraan hyytymän syntymiseen valtimon pinnalla ja sen kulkeutumiseen aivoihin, tai toisaalta rasva-aineiden pääsyyn valtimon seinämän sisälle kasvattaen ahtauman sisäistä rasvaydintä ja aiheuttaen haitallisia hapettumisprosesseja altistaen edelleen KVA:aa sisäisille mikroverenvuodoille. Viimeksimainitut tapahtumasarjat tekevät KVA:sta repeytymisherkän johtaen potilaan oirehtimiseen. Lisätutkimuksia vaaditaan vielä, jotta em. tekijöiden tarkempi osuus KVA:n muuntumisessa epävakaaksi saadaan selvitettyä, ja mahdollisesti KVA:n vakauttamiseen johtavia hoitoja tulevaisuudessa kehitettyä

MixFit : Methodology for Computing Ancestry-Related Genetic Scores at the Individual Level and Its Application to the Estonian and Finnish Population Studies

Ancestry information at the individual level can be a valuable resource for personalized medicine, medical, demographical and history research, as well as for tracing back personal history. We report a new method for quantitatively determining personal genetic ancestry based on genome-wide data. Numerical ancestry component scores are assigned to individuals based on comparisons with reference populations. These comparisons are conducted with an existing analytical pipeline making use of genotype phasing, similarity matrix computation and our addition-multidimensional best fitting by MixFit. The method is demonstrated by studying Estonian and Finnish populations in geographical context. We show the main differences in the genetic composition of these otherwise close European populations and how they have influenced each other. The components of our analytical pipeline are freely available computer programs and scripts one of which was developed in house.Peer reviewe

Ocular signs of carotid stenosis in ipsi- and contralateral eyes before and after carotid endarterectomy : a prospective study

Purpose We describe hypoperfusion-related and embolic ocular signs of carotid stenosis (CS) before and six months after carotid endarterectomy (CEA) in a CS population. Methods We enrolled prospectively 70 CEA patients (81% male, mean age 69) and 41 non-medicated control subjects (76%, 68), from March 2015 to December 2018, assessing intraocular pressure (IOP), best-corrected visual acuity (BCVA) in logMAR units and performing a bio-microscopy examination. Results Main index symptoms included amaurosis fugax (Afx) (29, 41%) and hemispheric TIA (17, 24%), and 17 (24%) were asymptomatic. Of the 70, 17 patients (24%, 95% CI 16-36) showed ocular signs of CS. Of four embolic (Hollenhorst plaques) findings, one small macular plaque disappeared postoperatively. Four had hypoperfusion, that is ocular ischaemic syndrome (OIS), requiring panretinal photocoagulation: one for multiple mid-peripheral haemorrhages, two for iris neovascularization and one for neovascular glaucoma (NVG); only the NVG proved irreversible. Nine (de novo in three) showed mild OIS, that is only few mid-peripheral haemorrhages, ranging pre- /postoperatively in ipsilateral eyes from one to eleven (median two)/ one to two (median one), and in contralateral eyes from three to nine (median five)/ one to six (median three). Pre- and postoperative median BCVA was 0 or better, and mean IOP was normal, except in the NVG patient. Temporary visual impairment from 0 to 0.3 occurred in one eye soon after CEA due to ocular hyperperfusion causing macular oedema. Conclusions Ocular signs of CS are common in CEA patients, ranging from few mid-peripheral haemorrhages to irreversible NVG. Clinicians should be aware of these signs in detecting CS.Peer reviewe

Subfoveal choroidal thickness in ipsi- and contralateral eyes of patients with carotid stenosis before and after carotid endarterectomy : a prospective study

Purpose To compare subfoveal choroidal thickness (SFCT) and associated clinical variables in patients with carotid stenosis (CS) before and 6 months after carotid endarterectomy (CEA). Methods The prospective non-randomized Helsinki Carotid Endarterectomy Study - Brain and Eye Sub-sTudy included seventy patients (81% male, mean age 69 years) and 40 control subjects (77% male, 68 years), from March 2015 to December 2018. Ophthalmological examination included SFCT measured with enhanced-depth imaging-optical coherence tomography. Carotid stenosis (CS) was more severe (>= 70% stenosis in 92%) ipsilateral to the CEA than contralaterally ( Results At baseline, patients had thinner mean SFCT than control subjects in both eyes (ipsilateral, 222 versus 257 mu m and contralateral, 217 versus 258 mu m, p Conclusions Subfoveal choroidal thickness (SFCT) is thinner in patients with CS without association between SFCT and the grade of CS. Unchanged SFCT after CEA suggests, that choroidal vessels in severe CS are unable to react to increased blood flow. Bilaterally thin SFCT could be considered as yet another sign of CS.Peer reviewe

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

Flicker-induced retinal vascular dilation in ipsi- and contralateral eyes of patients with carotid stenosis before and after carotid endarterectomy : a prospective study

Purpose Retinal vascular function was assessed in patients with carotid stenosis (CS) before and six months after carotid endarterectomy (CEA) and in controls at a six-month interval. Methods We studied 68 patients (81% male, mean age 69) and 41 healthy non-medicated controls (77%, 68) from March 2015 to December 2018. Our ophthalmological examination included flicker-induced arteriolar and venular measurements with a Dynamic Vessel Analyser in both eyes. Results At baseline, flicker-induced arteriolar and venular dilation was reduced in the ipsilateral eyes of the patients compared with dilation in the controls (arteriolar 1.0% versus 2.6%, p = 0.001 and venular 2.2% versus 2.8%, p = 0.049). These differences subsided after CEA. In patients' ipsilateral eyes, flicker-induced arteriolar dilation was borderline postoperatively (preoperative 1.0% versus postoperative 1.6%, p = 0.06), whereas venular dilation increased (2.2% versus 2.8%, p = 0.025). We found various tentative associations with the change in flicker-induced dilations after CEA, but not with the preoperative dilations. Conclusions Postoperative recovery of the reduced flicker-induced arteriolar and venular dilatation in the ipsilateral eye shows that, after CEA, the activity-dependent vascular reactivity of haemodynamically compromised retinal tissue can improve.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

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

The prevalence of metabolic syndrome and metabolically healthy obesity in Europe: a collaborative analysis of ten large cohort studies

Peer reviewe

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms