Smooth muscle cells in human atherosclerosis: proteomic profiling reveals differences in expression of Annexin A1 and mitochondrial proteins in carotid disease.

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Interleukin-6 and microRNA profiles induced by oral bacteria in human atheroma derived and healthy smooth muscle cells

Background Atherosclerosis is an inflammatory disease with possible contributions from bacterial antigens. We aimed to investigate the role of oral bacteria as inducers of inflammatory cascades in smooth muscle cells from carotid endarterectomy patients (AthSMCs) and healthy controls (HSMCs). Findings Inactivated Streptococcus mitis, S. sanguinis, S. gorgonii, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were used to stimulate inflammation in HSMCs and AthSMCs. Tumor necrosis factor-α (TNFα) was used as a positive control in all stimulations. Interleukin-6 (IL-6) levels were determined from cell culture supernatants and microRNA expression profiles from cells after 24 h of bacterial stimulation. Genome wide expression (GWE) analyses were performed after 5 h stimulation. All studied bacteria induced pro inflammatory IL-6 production in both SMCs. The most powerful inducer of IL-6 was A. actinomycetemcomitans (p < 0.001). Of the 84 studied miRNAs, expression of 9 miRNAs differed significantly (p ≤ 0.001) between HSMCs and AthSMCs stimulated with inactivated bacteria or TNFα. The data was divided into two groups: high IL-6 producers (A. actinomytectemcomititans and TNFα) and low IL-6 producers (streptococcal strains and P. gingivalis). The expression of 4 miRNAs (miR-181-5p, −186-5p, −28-5p and −155-5p) differed statistically significantly (p < 0.001) between healthy HSMCs and AthSMCs in the low IL-6 producer group. According to multidimensional scaling, two gene expression clusters were seen: one in HSMCs and one AthSMCs. Conclusions Our results suggest that inactivated oral bacteria induce inflammation that is differently regulated in healthy and atherosclerotic SMCs.BioMed Central/SpringerOpen Journa

hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver

Peer reviewe

Methylation status of nc886 epiallele reflects periconceptional conditions and is associated with glucose metabolism through nc886 RNAs

BackgroundNon-coding RNA 886 (nc886) is coded from a maternally inherited metastable epiallele. We set out to investigate the determinants and dynamics of the methylation pattern at the nc886 epiallele and how this methylation status associates with nc886 RNA expression. Furthermore, we investigated the associations between the nc886 methylation status or the levels of nc886 RNAs and metabolic traits in the YFS and KORA cohorts. The association between nc886 epiallele methylation and RNA expression was also validated in induced pluripotent stem cell (iPSC) lines.ResultsWe confirm that the methylation status of the nc886 epiallele is mostly binomial, with individuals displaying either a non- or hemi-methylated status, but we also describe intermediately and close to fully methylated individuals. We show that an individual's methylation status is associated with the mother's age and socioeconomic status, but not with the individual's own genetics. Once established, the methylation status of the nc886 epiallele remains stable for at least 25 years. This methylation status is strongly associated with the levels of nc886 non-coding RNAs in serum, blood, and iPSC lines. In addition, nc886 methylation status associates with glucose and insulin levels during adolescence but not with the indicators of glucose metabolism or the incidence of type 2 diabetes in adulthood. However, the nc886-3p RNA levels also associate with glucose metabolism in adulthood.ConclusionsThese results indicate that nc886 metastable epiallele methylation is tuned by the periconceptional conditions and it associates with glucose metabolism through the expression of the ncRNAs coded in the epiallele region.</p

Association and functional studies on promoter polymorphisms within the apolipoprotein E gene, regarding lipid metabolism and atherosclerosis

Ateroskleroosissa eli valtimonkovettumataudissa valtimon seinämään kertyy vähitellen rasvatäytteisiä vaahtosoluja, fibroottista soluväliainetta ja kalkkeutumia, jotka vähitellen muodostavat verisuonta tukkivan aterooman. Tämä on perusta sepel- ja aivovaltimoiden kovettumataudin kehittymiselle. Ateroskleroosin riskiä lisäävät monet eri tekijät kuten korkea ikä, miessukupuoli, korkea verenpaine, tupakointi, korkea LDL-kolesteroli ja eräät geneettiset tekijät kuten apolipoproteiini E geenin (APOE) ε4-alleeli. Apolipoproteiini E (apoE) on rasvojen kuljetukseen osallistuva proteiini, jota koodittavasta geenistä on löydetty useita muuntelevia kohtia eli polymorfioita. Tunnetuin näistä on ε2/ε3/ε-4-alleelien muuntelu, jonka seurauksena voi syntyä kuusi erilaista alleeli-yhdistelmää eli genotyyppiä. Yleisintä APOE genotyyppiä ε3/ε3 kantaa noin 60 % suomalaisista. Myös APOE geenin säätelyalueelta on tunnistettu useita polymorfioita, jotka voivat vaikuttaa esimerkiksi geenin luentaan ja sitä kautta apoE-proteiinin määrään verenkierrossa. Väitöskirjatyössä käytettiin kolmea kliinistä ja kahta ruumiinavausaineistoa, joista tutkittiin yhteensä yli 2500 henkilöä. Tarkoituksena oli selvittää assosiaatioanalyysin avulla, yhdistyvätkö APOE geenin säätelyalueen kaksi polymorfiaa -219G/T ja +113G/C seerumin lipoproteiinitasoihin, kaulavaltimoiden varhaisiin ateroskleroottisiin muutoksiin sekä aivoinfarkti- ja aivoateroskleroosiriskiin. Viimeisessä osatyössä tutkittiin myös APOE geeni-tupakointi interaktion yhteyttä sepelvaltimoiden ja vatsa-aortan rasvajuosteiden pinta-alaan. Työssä keskityttiin tutkimaan APOE geenin säätelyalueen polymorfioita kaikista yleisimmän APOE ε3/ε3-genotyyppiryhmän sisällä. Siten oli mahdollista keskittyä etsimään säätelyalueen polymorfioiden itsenäisiä vaikutuksia ja neutraloida ε2/ε3/ε4- muuntelun vaikutus. Haluttiin myös selvittää, vaikuttaako +113G/C polymorfia geeniluennan eli transkription tehokkuuteen ja sitooko alue mahdollisesti joitain transkriptioon vaikuttavia säätelyproteiineja eli transkriptiotekijöitä. Tulokset osoittivat, että tutkitut APOE geenin säätelyalueen polymorfiat yhdistyvät seerumin lipoproteiinitasoihin niin poikkileikkaus- kuin pitkittäistutkimuksessakin (21-vuotisseurantatutkimus) mutta ne eivät silti selittäneet kaulavaltimon varhaisia ateroskleroottisia muutoksia. Kuitenkin voitiin osoittaa, että -219G- ja +113G-alleelien kantajilla on kohonnut aivoinfarktiriski. Toisaalta miehillä, jotka kantoivat -219T- tai +113C-alleelia oli vaikeampi aivoateroskleroosi verrattuna -219G/G ja +113G/G genotyyppien kantajiin. Tutkimuksessa tuli esille mielenkiintoinen havainto APOE geeni-tupakointi-interaktiosta vatsa-aortan rasvajuosteiden suhteen: tiettyjen genotyyppiryhmien (-219G/G tai +113G/G) sisällä tupakoitsijoilla oli suuremmat vatsa-aortan rasvajuosteet kuin ei-tupakoitsijoilla. Nämä tulokset siis vahvistavat käsitystä, jonka mukaan monitekijäisissä sairauksissa kuten ateroskleroosissa, geenien ja ympäristön välinen monimutkainen vuorovaikutus on tärkeä tekijä taudin kehittymisessä. Pystyttiin myös osoittamaan, että +113G/C polymorfialla on vaikutusta geenitranskriptioon ja että se sitoo transkriptiotekijöitä, joista yhdeksi mahdolliseksi tunnistettiin RBP-Jκ.Background. Atherosclerosis is a complex disease in which lipid laden foam cells, fibrotic matrix and calcification gradually accumulate in the arterial wall. The disease begins already in childhood and progresses throughout life. Atherosclerosis can be asymptomatic for decades and develop clinical symptoms, such as heart attack or stroke, later in life. Many risk factors for atherosclerosis have been identified, including advanced age, male sex, high blood pressure, smoking, dyslipidemia and the ε4-allele of the apolipoprotein E gene (APOE). Objectives. This study is an attempt to clarify the role of the APOE promoter polymorphisms -219G/T and +113G/C and APOE haplotypes in explaining the variation of serum lipid, apolipoprotein and lipoprotein concentrations, as well as studying the possible association of these polymorphisms with the early markers of atherosclerosis, intracranial atherosclerosis as well as ischemic stroke, especially within the most common APOE ε3/ε3 genotype group. Furthermore, we wanted to test whether the alleles of the APOE +113G/C polymorphism have differential effects on APOE transcription. Subjects and methods. The study was based on five study series: three clinical and two autopsy studies. The first two clinical series altogether comprised 824 subjects (UKK and Cardiovascular in Young Finns Study). They were used to study the associations of the APOE promoter polymorphisms -219G/T and +113G/C and their haplotypes, with several variables of lipoprotein metabolism (I), as well as with longitudinal changes in cholesterol values (II) in apparently healthy Finns. The third clinical series consisting of 237 stroke cases and 326 controls, of Belgian origin, studied the association of the APOE polymorphisms/haplotypes with the risk of ischemic stroke (III). The Tampere Autopsy Study (TASTY, n = 604) was utilized to study the intracranial atherosclerosis within different APOE genotype groups (III). In the Helsinki Sudden Death Study (n = 700), the association of APOE promoter polymorphisms/haplotypes with fatty streak areas in two coronary arteries and the abdominal aorta were explored (IV). Finally, the luciferase assay was used to study the effect of the APOE +113G/C polymorphism on transcriptional efficiency in a human hepatoma cell line. Furthermore, the electrophoretic mobility shift assay (EMSA) was used to study nuclear protein binding to the APOE +113G/C region (IV). Results. Our results show that both APOE promoter polymorphisms -219G/T and +113G/C, as well as their haplotypes seem to affect various serum lipid/apolipoprotein concentrations, independent of the APOE ε2/ε3/ε4 genotype. The -219T/T and +113C/C-genotype carriers had lower very low-density (VLDL) cholesterol, apolipoprotein B (apoB) and triglyceride (TG) concentrations when compared to G/G carriers (I). The -219T/+113C/ε3 haplotype carriers showed higher low-density lipoprotein (LDL) cholesterol and total cholesterol values throughout the 21 year follow-up compared to non-carriers (II). We did not detect statistically significant differences in any of the studied subclinical markers of atherosclerosis (such as intima-media thickness), between the APOE promoter genotypes or haplotypes (II). Within the APOE ε3/ε3 carriers, there was a statistically significant promoter genotype-smoking interaction on the fatty streak area of the abdominal aorta (IV). Within non-smokers, -219T- and +113C-allele carriers had larger fatty streak areas in abdominal aorta compared to G/G genotype carriers. Moreover, carriers of haplotype -219T/+113C/ε3 had larger fatty streak areas in abdominal aorta compared to homozygous carriers of haplotype -219G/+113G/?3. In smokers the situation was opposite. The -219G- or +113G-allele carriers were at increased risk of ischemic stroke, and the -219T-allele carrier men had more severe intracranial atherosclerosis (III). Our functional studies revealed differences in the transcriptional activity of different APOE +113G/C alleles. The C-allele had a higher transcriptional activity compared to the G-allele. The EMSAs indicated that there is a quantitative difference in protein(s) from hepatic nuclear lysate binding to the G- and C-alleles: the G-allele bound nuclear protein(s) with higher affinity compared to the C-allele. A transcription factor RBP-Jκ was shown to bind with higher affinity to the C-allele compared to the G-allele (IV). Conclusions. The APOE promoter polymorphisms -219G/T and +113G/C associated with variations in serum lipid/apolipoprotein concentration and to some extent also predicted the risk of ischemic stroke. Additionally, our studies showed that the studied APOE promoter polymorphisms interact with smoking, an environmental risk factor, in defining the lesion areas in the abdominal aorta. Moreover, the +113G/C polymorphism was shown to be functional, the C-allele associating with a higher transcriptional activity when compared to the G-allele. The studied APOE promoter polymorphisms are important and functional regulators of lipid metabolism and can, together with smoking, affect the development of atherosclerosis

Association and functional studies on promoter polymorphisms within the apolipoprotein E gene, regarding lipid metabolism and atherosclerosis

Ateroskleroosissa eli valtimonkovettumataudissa valtimon seinämään kertyy vähitellen rasvatäytteisiä vaahtosoluja, fibroottista soluväliainetta ja kalkkeutumia, jotka vähitellen muodostavat verisuonta tukkivan aterooman. Tämä on perusta sepel- ja aivovaltimoiden kovettumataudin kehittymiselle. Ateroskleroosin riskiä lisäävät monet eri tekijät kuten korkea ikä, miessukupuoli, korkea verenpaine, tupakointi, korkea LDL-kolesteroli ja eräät geneettiset tekijät kuten apolipoproteiini E geenin (APOE) ε4-alleeli. Apolipoproteiini E (apoE) on rasvojen kuljetukseen osallistuva proteiini, jota koodittavasta geenistä on löydetty useita muuntelevia kohtia eli polymorfioita. Tunnetuin näistä on ε2/ε3/ε-4-alleelien muuntelu, jonka seurauksena voi syntyä kuusi erilaista alleeli-yhdistelmää eli genotyyppiä. Yleisintä APOE genotyyppiä ε3/ε3 kantaa noin 60 % suomalaisista. Myös APOE geenin säätelyalueelta on tunnistettu useita polymorfioita, jotka voivat vaikuttaa esimerkiksi geenin luentaan ja sitä kautta apoE-proteiinin määrään verenkierrossa. Väitöskirjatyössä käytettiin kolmea kliinistä ja kahta ruumiinavausaineistoa, joista tutkittiin yhteensä yli 2500 henkilöä. Tarkoituksena oli selvittää assosiaatioanalyysin avulla, yhdistyvätkö APOE geenin säätelyalueen kaksi polymorfiaa -219G/T ja +113G/C seerumin lipoproteiinitasoihin, kaulavaltimoiden varhaisiin ateroskleroottisiin muutoksiin sekä aivoinfarkti- ja aivoateroskleroosiriskiin. Viimeisessä osatyössä tutkittiin myös APOE geeni-tupakointi interaktion yhteyttä sepelvaltimoiden ja vatsa-aortan rasvajuosteiden pinta-alaan. Työssä keskityttiin tutkimaan APOE geenin säätelyalueen polymorfioita kaikista yleisimmän APOE ε3/ε3-genotyyppiryhmän sisällä. Siten oli mahdollista keskittyä etsimään säätelyalueen polymorfioiden itsenäisiä vaikutuksia ja neutraloida ε2/ε3/ε4- muuntelun vaikutus. Haluttiin myös selvittää, vaikuttaako +113G/C polymorfia geeniluennan eli transkription tehokkuuteen ja sitooko alue mahdollisesti joitain transkriptioon vaikuttavia säätelyproteiineja eli transkriptiotekijöitä. Tulokset osoittivat, että tutkitut APOE geenin säätelyalueen polymorfiat yhdistyvät seerumin lipoproteiinitasoihin niin poikkileikkaus- kuin pitkittäistutkimuksessakin (21-vuotisseurantatutkimus) mutta ne eivät silti selittäneet kaulavaltimon varhaisia ateroskleroottisia muutoksia. Kuitenkin voitiin osoittaa, että -219G- ja +113G-alleelien kantajilla on kohonnut aivoinfarktiriski. Toisaalta miehillä, jotka kantoivat -219T- tai +113C-alleelia oli vaikeampi aivoateroskleroosi verrattuna -219G/G ja +113G/G genotyyppien kantajiin. Tutkimuksessa tuli esille mielenkiintoinen havainto APOE geeni-tupakointi-interaktiosta vatsa-aortan rasvajuosteiden suhteen: tiettyjen genotyyppiryhmien (-219G/G tai +113G/G) sisällä tupakoitsijoilla oli suuremmat vatsa-aortan rasvajuosteet kuin ei-tupakoitsijoilla. Nämä tulokset siis vahvistavat käsitystä, jonka mukaan monitekijäisissä sairauksissa kuten ateroskleroosissa, geenien ja ympäristön välinen monimutkainen vuorovaikutus on tärkeä tekijä taudin kehittymisessä. Pystyttiin myös osoittamaan, että +113G/C polymorfialla on vaikutusta geenitranskriptioon ja että se sitoo transkriptiotekijöitä, joista yhdeksi mahdolliseksi tunnistettiin RBP-Jκ.Background. Atherosclerosis is a complex disease in which lipid laden foam cells, fibrotic matrix and calcification gradually accumulate in the arterial wall. The disease begins already in childhood and progresses throughout life. Atherosclerosis can be asymptomatic for decades and develop clinical symptoms, such as heart attack or stroke, later in life. Many risk factors for atherosclerosis have been identified, including advanced age, male sex, high blood pressure, smoking, dyslipidemia and the ε4-allele of the apolipoprotein E gene (APOE). Objectives. This study is an attempt to clarify the role of the APOE promoter polymorphisms -219G/T and +113G/C and APOE haplotypes in explaining the variation of serum lipid, apolipoprotein and lipoprotein concentrations, as well as studying the possible association of these polymorphisms with the early markers of atherosclerosis, intracranial atherosclerosis as well as ischemic stroke, especially within the most common APOE ε3/ε3 genotype group. Furthermore, we wanted to test whether the alleles of the APOE +113G/C polymorphism have differential effects on APOE transcription. Subjects and methods. The study was based on five study series: three clinical and two autopsy studies. The first two clinical series altogether comprised 824 subjects (UKK and Cardiovascular in Young Finns Study). They were used to study the associations of the APOE promoter polymorphisms -219G/T and +113G/C and their haplotypes, with several variables of lipoprotein metabolism (I), as well as with longitudinal changes in cholesterol values (II) in apparently healthy Finns. The third clinical series consisting of 237 stroke cases and 326 controls, of Belgian origin, studied the association of the APOE polymorphisms/haplotypes with the risk of ischemic stroke (III). The Tampere Autopsy Study (TASTY, n = 604) was utilized to study the intracranial atherosclerosis within different APOE genotype groups (III). In the Helsinki Sudden Death Study (n = 700), the association of APOE promoter polymorphisms/haplotypes with fatty streak areas in two coronary arteries and the abdominal aorta were explored (IV). Finally, the luciferase assay was used to study the effect of the APOE +113G/C polymorphism on transcriptional efficiency in a human hepatoma cell line. Furthermore, the electrophoretic mobility shift assay (EMSA) was used to study nuclear protein binding to the APOE +113G/C region (IV). Results. Our results show that both APOE promoter polymorphisms -219G/T and +113G/C, as well as their haplotypes seem to affect various serum lipid/apolipoprotein concentrations, independent of the APOE ε2/ε3/ε4 genotype. The -219T/T and +113C/C-genotype carriers had lower very low-density (VLDL) cholesterol, apolipoprotein B (apoB) and triglyceride (TG) concentrations when compared to G/G carriers (I). The -219T/+113C/ε3 haplotype carriers showed higher low-density lipoprotein (LDL) cholesterol and total cholesterol values throughout the 21 year follow-up compared to non-carriers (II). We did not detect statistically significant differences in any of the studied subclinical markers of atherosclerosis (such as intima-media thickness), between the APOE promoter genotypes or haplotypes (II). Within the APOE ε3/ε3 carriers, there was a statistically significant promoter genotype-smoking interaction on the fatty streak area of the abdominal aorta (IV). Within non-smokers, -219T- and +113C-allele carriers had larger fatty streak areas in abdominal aorta compared to G/G genotype carriers. Moreover, carriers of haplotype -219T/+113C/ε3 had larger fatty streak areas in abdominal aorta compared to homozygous carriers of haplotype -219G/+113G/?3. In smokers the situation was opposite. The -219G- or +113G-allele carriers were at increased risk of ischemic stroke, and the -219T-allele carrier men had more severe intracranial atherosclerosis (III). Our functional studies revealed differences in the transcriptional activity of different APOE +113G/C alleles. The C-allele had a higher transcriptional activity compared to the G-allele. The EMSAs indicated that there is a quantitative difference in protein(s) from hepatic nuclear lysate binding to the G- and C-alleles: the G-allele bound nuclear protein(s) with higher affinity compared to the C-allele. A transcription factor RBP-Jκ was shown to bind with higher affinity to the C-allele compared to the G-allele (IV). Conclusions. The APOE promoter polymorphisms -219G/T and +113G/C associated with variations in serum lipid/apolipoprotein concentration and to some extent also predicted the risk of ischemic stroke. Additionally, our studies showed that the studied APOE promoter polymorphisms interact with smoking, an environmental risk factor, in defining the lesion areas in the abdominal aorta. Moreover, the +113G/C polymorphism was shown to be functional, the C-allele associating with a higher transcriptional activity when compared to the G-allele. The studied APOE promoter polymorphisms are important and functional regulators of lipid metabolism and can, together with smoking, affect the development of atherosclerosis

Finnish Secondary Students’ Mental Models of Magnetism

We examined Finnish lower secondary students’ mental models of magnetism through their drawings, written explanations and interviews. Secondary students in Finland (N=12) engaged in six lessons designed specifically to target three key concepts in understanding magnetism: structure and organization (magnetic domains), magnetic fields and magnetic interactions. We describe how, with a finite number of key concepts introduced, students reflected upon and revised their mental models of magnetism and magnetic interactions towards more sophisticated and normative scientific views. We found two new categories of students’ models: the pole model and pole/field model. The critical moments in evolving the models happened during the investigations regarding understanding magnetic fields and magnetic internal structure. This article gives an example for teachers and researchers of how to follow students’ development of mental models in science.peerReviewe