Extracellular lipid particles in atherosclerosis and aortic stenosis

Aortic stenosis and athrosclerosis are slowly progressing diseases. Being clinically silent, as they start developing decades before they cause symptoms, cardiovascular diseases and atherosclerosis in particular, are the leading cause of morbitidy and mortality in the Western world. Lipid accumulation begins both in the artery walls and in the aortic valves before any clinical signs of atherosclerosis or aortic stenosis can be detected. While atherosclerotic lesions are characterized with cells filled with lipid droplets i.e. foam cells, and they are found to be calcified only in the late stages of atherosclerosis, the stenotic aortic valve leaflets contain both lipid droplets and calcified nodules already in early lesions. The proteoglycan matrix common to artery wall and aortic valve leaflets retains the entering lipoprotein particles that are then enzymatically and oxidatively modified. Such modifications have an ability to transform the non-inflammatory plasma lipoprotein particles into crystals and particles that can induce sterile inflammation in the various intimal and valvular cells. This thesis was set to study the distribution and characteristics of the extracellular lipid and to reveal the origin of the extracellular lipid particles. Prior to the analyses of the isolated extracellular lipid particles, the human carotid artery plaques were imaged with three-dimensional electron microscopy and sections of human coronary arteries were analyzed with imaging mass spectrometry to study the spatial distribution of lipids in different stages of atherosclerosis. In the human coronary arteries, the lipid domains found in advanced atherosclerotic lesions were different from the domains found in atheroma-stage artery sections, and even more different from healthy sections of coronary arteries. In the human carotid artery plaques, cholesterol crystals were found to be large sheets or needle-like structures, and they appeared to be growing out from large lipid particles in the intima. For the purpose of studying the chemical and physical characteristics of the extracellular lipid particles, the extracellular particles were isolated from aortic valve leaflets and coronary artery plaques. The lipid particles were examined with multiple tools to study their lipid composition, protein composition, protein structure, size, and density. The extracellular lipid particles, both in human aortic valve leaflets and human carotid arteries, were found to be derived from plasma lipoproteins, mainly from low density lipoprotein (LDL) or very low density lipoprotein (VLDL). Apart from multiple small exchangeable lipoproteins, like apolipoprotein (apo) E, apoA-family, and apoC-family, the particles contained mainly apolipoprotein B-100 (apoB-100), the integral protein of LDL and VLDL, and they contained features which suggest that they were multiply modified. Another goal was to find which modifications would change the intimal or valvular extracellular lipid particles to such fused and aggregated lipid particles that were found in the valves and intimal plaques, and which modifications would induce a sterile inflammatory response similar to the response the extracellular lipid particles induce. To study the possible culprit modifications, both the extracellular lipid particles, in vitro-generated cholesterol crystals, and LDL that was modified by lipolysis, proteolysis, and oxidation, were applied to human primary monocyte-derived macrophages. Both the isolated extracellular lipid particles and LDL modified with a combination of phospholipolysis by PLA2 and cholesterol esterase were found to be able to activate a multiprotein complex inflammasome in human primary monocyte-derived macrophages in vitro, and to induce the secretion of proinflammatory cytokines. According to the results of this thesis, the lipid particles in the arterial intima and in the aortic valve are active components of atherosclerosis and aortic stenosis. They can induce the cells in the intima and in the valve to produce inflammatory cytokines and thus can affect the progress of these diseases.Aorttastenoosi eli aorttaläpän kalkkinen kovettumatauti ja ateroskleroosi eli valtimokovettumatauti ovat hitaasti eteneviä tauteja. Koska nämä sydän- ja verisuonitaudit kehittyvät piilossa oireita aiheuttamatta jopa vuosikymmenten ajan, ne ovat yhä edelleen suomalaisten ja läntisen maailman kuolinsyytilastojen kärjessä. Lipidit eli rasva-aineet alkavat kertyä sekä aorttaläppään että valtimoiden seinämään jo paljon ennen kuin mitään kliinisiä merkkejä on havaittavissa. Ateroskleroosissa plakit ovat tyypillisesti täynnä rasvapartikkeleita eli lipidipartikkeleita ja soluja, jotka ovat täynnä rasvapisaroita (vaahtosoluja) ja ne kalkkiintuvat vasta taudin myöhäisvaiheessa. Aorttaläpissä on sen sijaan yhtä aikaa sekä lipidipartikkeleita ja kiteytynyttä kalkkia. Sekä aorttaläpässä että valtimon seinämässä on proteoglykaanikerros, johon lipidipartikkelit tarttuvat kiinni. Kiinni tarttuneet lipidipartikkelit voivat voivat muokkaantua entsyymien vaikutuksesta tai hapettumisen seurauksena. lipidipartikkelien muokkaantuminen voi saada aorttaläpän tai valtimon sisäkerroksen solut reagoimaan steriilillä tulehdusreaktiolla tai muokkaantuneet lipidipartikkelien sisältämä kolesteroli voi kiteytyä suuriksi kolesterolikiteiksi. Tässä väitöskirjassa tutkittiin solunulkoisten lipidipartikkeleiden ominaisuuksia ja niiden levittäytymistä solujen väliseen tilaan. Siten pyrittiin selvittämään solunulkoisten lipidipartikkeleiden alkuperä. Ihmisen kaulavaltimoiden seinämästä valmistettiin malleja kolmiulotteisten elektronimikroskooppikuvien avulla ja ihmisen sepelvaltimoleikkeitä tutkittiin kuvantavalla massaspektrometrilla. Näin saatiin selville, kuinka lipidipartikkelit sijaitsivat valtimoiden seinämässä. Terveiden sepelvaltimoiden lipidipartikkelikertymien laatu ja paikka erosivat aterooma-vaiheen plakkien lipidipartikkelikertymistä ja vielä enemmän ne erosivat pitkälle edenneestä ateroskleroottisesta plakista. Ihmisen kaulavaltimoiden plakeissa kolesterolikiteet olivat sekä neulamaisia että suuria levyjä ja ne näyttivät kasvavan suoraan suurista lipidipartikkeleista. Kemiallisten ja fysikaalisten ominaisuuksien selvittämiseksi lipidipartikkelit eristettiin sekä aorttaläpistä että kaulavaltimoista. Niistä tutkittiin lipidikoostumusta, proteiinikoostumusta, proteiiniosan rakennetta, partikkelien kokoa ja tiheyttä. Sekä aorttaläpän että kaulavaltimon lipidipartikkeleiden havaittiin olevan peräisin plasman apolipoproteiini (apo)B-100:aa sisältävistä lipoproteiinipartikkeleista, todennäköisimmin joko kevyen lipoproteiinin (LDL) tai erittäin kevyen lipoproteiinin (VLDL) partikkeleista. Partikkelit sisälsivät apoB-100:n lisäksi myös pieniä vaihdettavia apolipoproteiineja, kuten apolipoproteiini (apo) E:tä, apoA-proteiiniperheen ja apoC-proteiiniperheen apolipoproteiineja. ApoB-100 (LDL:n ja VLDL:n tärkein rakenneproteiini) oli yleisin löydetty proteiini. Lipidipartikkeleissa nähtiin merkkejä moninkertaisesta muokkautumisesta. Tutkimuksen toisena tavoitteena oli saada selville, miten aorttaläpän ja valtimon sisäseinämään jääneet lipidipartikkelit muuntuvat plasman lipoproteiineista suuriksi fuusioituneiksi ja aggregoituneiksi lipidipartikkeleiksi. Lisäksi haluttiin tietää, mitkä muokkausmekanismit muuntaisivat lipoproteiinit niin, että solut reagoisivat steriilillä tulehdusreaktiolla eli inflammasomi-kompleksin aktivaatiolla. Mahdollisia muuntumismekanismeja tutkittiin muokkaamalla LDL-partikkeleita lipolyyttisillä ja proteolyyttisillä entsyymeillä ja hapettamalla niitä. Näin muokattuja LDL-partikkeleita ja kaulavaltimoista eristettyjä lipidipartikkeleita annettiin ihmisen plasmasta eristetyistä monosyyteistä erilaistetuille makrofageille. Sekä solunulkoiset lipidipartikkelit että fosfolipolyysillä muokatut plasman lipoproteiinit saivat koeolosuhteissa makrofagit reagoimaan inflammasomin aktivaatiolla ja tuottamaan tulehdusvälittäjäaineita. Väitöskirjan tulosten mukaan voidaan todeta, että lipidipartikkelit aorttaläpässä ja valtimon sisäseinämässä ovat oleellinen osa aorttastenoosin ja ateroskleroosin syntyprosessissa. Ne saavat aorttaläpän ja valtimon sisäseinämän solut tuottamaan tulehdusvälittäjäaineita ja vaikuttavat siten sekä aorttastenoosin että ateroskleroosin etenemiseen

Molecular and Cellular Markers in Skeletal Muscle Damage after Acute Voluntary Exercise Containing Eccentric Muscle Contractions

In eccentric muscle contraction, the muscle is lengthening while contracting. For example, in downhill walking, the thigh muscles are contracting eccentrically. It is well known that unaccustomed eccentric exercise causes pain and may lead to inflammation reactions on muscles few days after the exercise. The theme of the present chapter is molecular and cellular markers in skeletal muscle damage after voluntary exercise containing eccentric muscle contractions. The chapter contains three topics: In the first topic, the damaging process followed by regeneration is demonstrated with antibody stainings of connective tissue, plasma membrane, and cytoskeletal proteins. The second topic is infiltration of inflammatory cells in damaged skeletal muscle. Neutrophils are usually the first inflammatory cells mostly present in the injured tissues; however, neutrophils are not present in exercise-induced skeletal muscle damage. Finally, the relationship between skeletal muscle damage and systematic markers, serum creatine kinase and voluntary maximal force production, is described

Lymphatic vessels are present in human saccular intracranial aneurysms

Saccular intracranial aneurysm (sIA) rupture leads to subarachnoid haemorrhage and is preceded by chronic inflammation and atherosclerotic changes of the sIA wall. Increased lymphangiogenesis has been detected in atherosclerotic extracranial arteries and in abdominal aortic aneurysms, but the presence of lymphatic vessels in sIAs has remained unexplored. Here we studied the presence of lymphatic vessels in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), using immunohistochemical and immunofluorescence stainings for lymphatic endothelial cell (LEC) markers. Of these LEC-markers, both extracellular and intracellular LYVE-1-, podoplanin-, VEGFR-3-, and Prox1-positive stainings were detected in 83%, 94%, 100%, and 72% of the 36 sIA walls, respectively. Lymphatic vessels were identified as ring-shaped structures positive for one or more of the LEC markers. Of the sIAs, 78% contained lymphatic vessels positive for at least one LEC marker. The presence of LECs and lymphatic vessels were associated with the number of CD68+ and CD163+ cells in the sIA walls, and with the expression of inflammation indicators such as serum amyloid A, myeloperoxidase, and cyclo-oxygenase 2, with the presence of a thrombus, and with the sIA wall rupture. Large areas of VEGFR-3 and alpha-smooth muscle actin (alpha SMA) double-positive cells were detected in medial parts of the sIA walls. Also, a few podoplanin and alpha SMA double-positive cells were discovered. In addition, LYVE-1 and CD68 double-positive cells were detected in the sIA walls and in the thrombus revealing that certain CD68+ macrophages are capable of expressing LEC markers. This study demonstrates for the first time the presence of lymphatic vessels in human sIA walls. Further studies are needed to understand the role of lymphatic vessels in the pathogenesis of sIA.Peer reviewe

Myeloperoxidase Associates With Degenerative Remodeling and Rupture of the Saccular Intracranial Aneurysm Wall

Rupture of a saccular intracranial aneurysm (sIA) is often fatal. Thus, early detection of rupture-prone sIAs is vital. Myeloperoxidase (MPO), derived mainly from neutrophils, associates with sIA rupture, and therefore its role in sIA pathogenesis warrants further studies. We analyzed MPO and its association with other histological markers in 36 (16 unruptured and 20 ruptured) sIA samples by immunohistochemistry. MPO was present in all studied sIAs, and its expression associated with wall inflammatory cell infiltrations (r = 0.50, 0.63, and 0.75, all p <0.002), degenerative remodeling (p = 0.002) and rupture (p = 0.003). MPO associated strongly with the presence of organized luminal thrombi (p <0.001), which also stained positive for MPO. Polymorphonuclear MPO+ cells were detected in the sIA walls, indicating neutrophils as MPO-source. MPO correlated strongly with accumulation of oxidized lipids (r = 0.67, p <0.001) and loss of smooth muscle cells (r = -0.68, p <0.001), suggesting that MPO is a relevant source of oxidative stress leading to cell death in the sIA wall. Furthermore, MPO associated with erythrocyte fragmentation (r = 0.74, p <0.001) and iron deposition (p = 0.041), 2 outcomes known to amplify MPO-dependent oxidative stress. Taken together, these results suggest that MPO associates with degenerative remodeling predisposing to sIA wall rupture and may serve as a biomarker of a rupture-prone sIA wall.Peer reviewe

Serum Amyloid A Is Present in Human Saccular Intracranial Aneurysm Walls and Associates With Aneurysm Rupture

Saccular intracranial aneurysm (sIA) rupture leads to a disabling subarachnoid hemorrhage. Chronic inflammation and lipid accumulation in the sIA wall contribute to wall degenerative remodeling that precedes its rupture. A better understanding of the pathobiological process is essential for improved future treatment of patients carrying sIAs. Serum amyloid A (SAA) is an acute-phase protein produced in response to acute and chronic inflammation and tissue damage. Here, we studied the presence and the potential role of SAA in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), that had previously been studied by histology and immunohistochemistry. SAA was present in all sIAs, but the extent of immunopositivity varied greatly. SAA immunopositivity correlated with wall degeneration (p=0.028) and rupture (p=0.004), with numbers of CD163-positive and CD68-positive macrophages and CD3-positive T lymphocytes (all pPeer reviewe

Modified Lipoprotein-Derived Lipid Particles Accumulate in Human Stenotic Aortic Valves

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Prebiotic Xylo-Oligosaccharides Ameliorate High-Fat-Diet-Induced Hepatic Steatosis in Rats

Understanding the importance of the gut microbiota (GM) in non-alcoholic fatty liver disease (NAFLD) has raised the hope for therapeutic microbes. We have shown that high hepatic fat content associated with low abundance of Faecalibacterium prausnitzii in humans and, further, the administration of F. prausnitzii prevented NAFLD in mice. Here, we aimed at targeting F. prausnitzii by prebiotic xylo-oligosaccharides (XOS) to treat NAFLD. First, the effect of XOS on F. prausnitzii growth was assessed in vitro. Then, XOS was supplemented or not with high (HFD, 60% of energy from fat) or low (LFD) fat diet for 12 weeks in Wistar rats (n = 10/group). XOS increased F. prausnitzii growth, having only a minor impact on the GM composition. When supplemented with HFD, XOS ameliorated hepatic steatosis. The underlying mechanisms involved enhanced hepatic β-oxidation and mitochondrial respiration. Nuclear magnetic resonance (1H-NMR) analysis of cecal metabolites showed that, compared to the HFD, the LFD group had a healthier cecal short-chain fatty acid profile and on the HFD, XOS reduced cecal isovalerate and tyrosine, metabolites previously linked to NAFLD. Cecal branched-chain fatty acids associated positively and butyrate negatively with hepatic triglycerides. In conclusion, XOS supplementation can ameliorate NAFLD by improving hepatic oxidative metabolism and affecting GM

Prebiotic Xylo-Oligosaccharides Ameliorate High-Fat-Diet-Induced Hepatic Steatosis in Rats

Understanding the importance of the gut microbiota (GM) in non-alcoholic fatty liver disease (NAFLD) has raised the hope for therapeutic microbes. We have shown that high hepatic fat content associated with low abundance of Faecalibacterium prausnitzii in humans and, further, the administration of F. prausnitzii prevented NAFLD in mice. Here, we aimed at targeting F. prausnitzii by prebiotic xylo-oligosaccharides (XOS) to treat NAFLD. First, the effect of XOS on F. prausnitzii growth was assessed in vitro. Then, XOS was supplemented or not with high (HFD, 60% of energy from fat) or low (LFD) fat diet for 12 weeks in Wistar rats (n = 10/group). XOS increased F. prausnitzii growth, having only a minor impact on the GM composition. When supplemented with HFD, XOS ameliorated hepatic steatosis. The underlying mechanisms involved enhanced hepatic β-oxidation and mitochondrial respiration. Nuclear magnetic resonance (1H-NMR) analysis of cecal metabolites showed that, compared to the HFD, the LFD group had a healthier cecal short-chain fatty acid profile and on the HFD, XOS reduced cecal isovalerate and tyrosine, metabolites previously linked to NAFLD. Cecal branched-chain fatty acids associated positively and butyrate negatively with hepatic triglycerides. In conclusion, XOS supplementation can ameliorate NAFLD by improving hepatic oxidative metabolism and affecting GM

Joint effects of alcohol use, smoking and body mass index as an explanation for the alcohol harm paradox : causal mediation analysis of eight cohort studies

Background and aims Lower socio-economic status (SES) is associated with higher alcohol-related harm despite lower levels of alcohol use. Differential vulnerability due to joint effects of behavioural risk factors is one potential explanation for this 'alcohol harm paradox'. We analysed to what extent socio-economic inequalities in alcohol-mortality are mediated by alcohol, smoking and body mass index (BMI), and their joint effects with each other and with SES. DesignCohort study of eight health examination surveys (1978-2007) linked to mortality data. Setting Finland.ParticipantsA total of 53 632 Finnish residents aged 25+ years.MeasurementsThe primary outcome was alcohol-attributable mortality. We used income as an indicator of SES. We assessed the joint effects between income and mediators (alcohol use, smoking and BMI) and between the mediators, adjusting for socio-demographic indicators. We used causal mediation analysis to calculate the total, direct, indirect and mediated interactive effects using Aalen's additive hazards models. Findings During 1 085 839 person-years of follow-up, we identified 865 alcohol-attributable deaths. We found joint effects for income and alcohol use and income and smoking, resulting in 46.8 and 11.4 extra deaths due to the interaction per 10 000 person-years. No interactions were observed for income and BMI or between alcohol and other mediators. The lowest compared with the highest income quintile was associated with 5.5 additional alcohol deaths per 10 000 person-years (95% confidence interval = 3.7, 7.3) after adjusting for confounders. The proportion mediated by alcohol use was negative (-69.3%), consistent with the alcohol harm paradox. The proportion mediated by smoking and BMI and their additive interactions with income explained 18.1% of the total effect of income on alcohol-attributable mortality. Conclusions People of lower socio-economic status appear to be more vulnerable to the effects of alcohol use and smoking on alcohol-attributable mortality. Behavioural risk factors and their joint effects with income may explain part of the alcohol harm paradox.Peer reviewe

Somatic MED12 Nonsense Mutation Escapes mRNA Decay and Reveals a Motif Required for Nuclear Entry

MED12 is a key component of the transcription-regulating Mediator complex. Specific missense and in-frame insertion/deletion mutations in exons 1 and 2 have been identified in uterine leiomyomas, breast tumors, and chronic lymphocytic leukemia. Here, we characterize the first MED12 5 end nonsense mutation (c.97G > T, p.E33X) identified in acute lymphoblastic leukemia and show that it escapes nonsense-mediated mRNA decay (NMD) by using an alternative translation initiation site. The resulting N-terminally truncated protein is unable to enter the nucleus due to the lack of identified nuclear localization signal (NLS). The absence of NLS prevents the mutant MED12 protein to be recognized by importin- and subsequent loading into the nuclear pore complex. Due to this mislocalization, all interactions between the MED12 mutant and other Mediator components are lost. Our findings provide new mechanistic insights into the MED12 functions and indicate that somatic nonsense mutations in early exons may avoid NMD. (C) 2017 Wiley Periodicals, Inc.Peer reviewe