Actin Dynamics in Muscle Cells

In every cell, actin is a key component involved in migration, cytokinesis, endocytosis and generation of contraction. In non-muscle cells, actin filaments are very dynamic and regulated by an array of proteins that interact with actin filaments and/or monomeric actin. Interestingly, in non-muscle cells the barbed ends of the filaments are the predominant assembly place, whereas in muscle cells actin dynamics was reported to predominate at the pointed ends of thin filaments. The actin-based thin filament pointed (slow growing) ends extend towards the middle of the sarcomere's M-line where they interact with the thick filaments to generate contraction. The actin filaments in muscle cells are organized into a nearly crystalline array and are believed to be significantly less dynamic than the ones in other cell types. However, the exact mechanisms of the sarcomere assembly and turnover are largely unknown. Interestingly, although sarcomeric actin structures are believed to be relatively non-dynamic, many proteins promoting actin dynamics are expressed also in muscle cells (e.g ADF/cofilin, cyclase-associated protein and twinfilin). Thus, it is possible that the muscle-specific isoforms of these proteins promote actin dynamics differently from their non-muscle counterparts, or that actin filaments in muscle cells are more dynamic than previously thought. To study protein dynamics in live muscle cells, I used primary cell cultures of rat cardiomyocytes. My studies revealed that a subset of actin filaments in cardiomyocyte sarcomeres displays rapid turnover. Importantly, I discovered that the turnover of actin filaments depends on contractility of the cardiomyocytes and that the contractility-induced actin dynamics plays an important role in sarcomere maturation. Together with previous studies those findings suggest that sarcomeres undergo two types of actin dynamics: (1) contractility-dependent turnover of whole filaments and (2) regulatory pointed end monomer exchange to maintain correct thin filament length. Studies involving an actin polymerization inhibitor suggest that the dynamic actin filament pool identified here is composed of filaments that do not contribute to contractility. Additionally, I provided evidence that ADF/cofilins, together with myosin-induced contractility, are required to disassemble non-productive filaments in developing cardiomyocytes. In addition, during these studies we learned that isoforms of actin monomer binding protein twinfilin, Twf-1 and Twf-2a localise to myofibrils in cardiomyocytes and may thus contribute to actin dynamics in myofibrils. Finally, in collaboration with Roberto Dominguez s laboratory we characterized a new actin nucleator in muscle cells - leiomodin (Lmod). Lmod localises towards actin filament pointed ends and its depletion by siRNA leads to severe sarcomere abnormalities in cardiomyocytes. The actin filament nucleation activity of Lmod is enhanced by interactions with tropomyosin. We also revealed that Lmod expression correlates with the maturation of myofibrils, and that it associates with sarcomeres only at relatively late stages of myofibrillogenesis. Thus, Lmod is unlikely to play an important role in myofibril formation, but rather might be involved in the second step of the filament arrangement and/or maintenance through its ability to promote tropomyosin-induced actin filament nucleation occurring at the filament pointed ends. The results of these studies provide valuable new information about the molecular mechanisms underlying muscle sarcomere assembly and turnover. These data offer important clues to understanding certain physiological and pathological behaviours of muscle cells. Better understanding of the processes occurring in muscles might help to find strategies for determining, diagnosis, prognosis and therapy in heart and skeletal muscles diseases.

Distribution and adhesion-promoting activity of alpha4 and alpha5 chain laminins in human endothelia and carcinomas

Basement membranes are specialized sheets of extracellular matrix found in contact with epithelia, endothelia, and certain isolated cells. They support tissue architecture and regulate cell behaviour. Laminins are among the main constituents of basement membranes. Due to differences between laminin isoforms, laminins confer structural and functional diversity to basement membranes. The first aim of this study was to gain insights into the potential functions of the then least characterized laminins, alpha4 chain laminins, by evaluating their distribution in human tissues. We thus created a monoclonal antibody specific for laminin alpha4 chain. By immunohistochemistry, alpha4 chain laminins were primarily localized to basement membranes of blood vessel endothelia, skeletal, heart, and smooth muscle cells, nerves, and adipocytes. In addition, alpha4 chain laminins were found in the region of certain epithelial basement membranes in the epidermis, salivary gland, pancreas, esophagus, stomach, intestine, and kidney. Because of the consistent presence of alpha4 chain laminins in endothelial basement membranes of blood vessels, we evaluated the potential roles of endothelial laminins in blood vessels, lymphatic vessels, and carcinomas. Human endothelial cells produced alpha4 and alpha5 chain laminins. In quantitative and morphological adhesion assays, human endothelial cells barely adhered to alpha4 chain-containing laminin-411. The weak interaction of endothelial cells with laminin-411 appeared to be mediated by alpha6beta1 integrin. The alpha5 chain-containing laminin-511 promoted endothelial cell adhesion better than laminin-411, but it did not promote the formation of cell-extracellular matrix adhesion complexes. The adhesion of endothelial cells to laminin-511 appeared to be mediated by Lutheran glycoprotein together with beta1 and alphavbeta3 integrins. The results suggest that these laminins may induce a migratory phenotype in endothelial cells. In lymphatic capillaries, endothelial basement membranes showed immunoreactivity for laminin alpha4, beta1, beta2, and gamma1 chains, type IV and XVIII collagens, and nidogen-1. Considering the assumed inability of alpha4 chain laminins to polymerize and to promote basement membrane assembly, the findings may in part explain the incomplete basement membrane formation in these vessels. Lymphatic capillaries of ovarian carcinomas showed immunoreactivity also for laminin alpha5 chain and its receptor Lutheran glycoprotein, emphasizing a difference between normal and ovarian carcinoma lymphatic capillaries. In renal cell carcinomas, immunoreactivity for laminin alpha4 chain was found in stroma and basement membranes of blood vessels. In most tumours, immunoreactivity for laminin alpha4 chain was also observed in the basement membrane region of tumour cell islets. Renal carcinoma cells produced alpha4 chain laminins. Laminin-411 did not promote adhesion of renal carcinoma cells, but inhibited their adhesion to fibronectin. Renal carcinoma cells migrated more on laminin-411 than on fibronectin. The results suggest that alpha4 chain laminins have a counteradhesive function, and may thus have a role in detachment and invasion of renal carcinoma cells.

LKB1 tumor suppression in Peutz-Jeghers Syndrome

Kasvainten, ajatellaan syntyvän yksittäisen solun perimän mutaatioista, jonka seurauksena tuon solun kasvu häiriintyy. Ruoansulatuskanavan polyyppien syntyä käytetään usein mallina siitä, miten nämä epiteelisoluun kerääntyvät mutaatiot aiheuttavat asteittain pahenevan kasvuhäiriön. Peutz–Jeghersin oireyhtymä (PJS) on perinnöllinen polypoosisyndrooma, jossa oireita aiheuttavat erityisesti maha-suolikanavan hamartomatoottiset polyypit. Noin puolella PJS potilaista havaitaan mutaatioita LKB1 kasvunrajoite geenissä. Hiirille joilta toinen Lkb1 alleeli on poistettu (Lkb1+/-) kehittyy PJS-tyypin maha-suolikanavan polyyppeja, joissa on epiteelin liikakasvun lisäksi merkittävä sileälihaskomponentti, aivan kuten PJS polyypeissa. Kuten myös muissa ruoansulatuskanavan polypooseissa, sekä PJS että hiirten polyypeissa Cyclo-oxygenaasi-2:n (COX-2) määrä on usein kohonnut. PJS-polyyppien kehittymisen molekulaarinen mekanismi on kuitenkin selvittämättä. Koska vain osa PJS potilaista kantaa LKB1 mutaatioita, mutaatiot jossakin toisessa lokuksessa saattaisivat selittää osan PJS tapauksista. Jotta PJS:n geneettinen tausta selviäisi, seulottiin kolmen LKB1:n kanssa interaktoivan proteiinin (BRG1, STRADα ja MO25α) geenit PJS potilaista joilla ei ole havaittu LKB1 mutaatioita. Yhdessäkään tutkituista geeneistä ei havaittu tautia aiheuttavia mutaatioita. Näiden kolmen geenin pois sulkeminen, ja uusien menetelmien ansiosta kasvanut havaittujen Lkb1 mutaatioden määrä viittaavat LKB1:n olevan useimpien PJS tapausten taustalla. COX-2:n estäjien käyttö on tehokkaasti vähentänyt polyyppien määrää familiaarisessa adenomatoottisessa polypoosissa. Tästä johtuen COX-2:n eston tehokkuutta tutkittiin PJS polypoosissa. PJS-tyypin polypoosin havaittin pienenevän merkittävästi Lkb1+/- hiirissä, joilta oli lisäksi poistettu toinen tai molemmat COX-2:n alleeleista. Lisäksi farmakologinen COX-2:n esto Celecoxib:lla vähensi polypoosia tehokkaasti. Näin ollen COX-2:n eston tehokkuutta tutkittiin seuraavaksi PJS potilaissa. Kuuden kuukauden Celecoxib hoidon jälkeen polypoosin havaittiin vähentyneen merkittävästi osalla potilaista (2/6). Nämä tulokset osoittavat COX-2:n roolin PJS-polyyppien kehityksessä, ja viittaavat COX-2:n eston vähentävän polypoosia. Kasvunrajoitegeenin klassisen määritelmän mukaan kasvaimen kehitys vaatii perinnöllisen mutaation lisäksi geenin toisenkin alleelin mutaation, mutta PJS-polyyppien häiriintyneestä epiteelistä ei kuitenkaan systemaattisesti löydy toista LKB1:n mutaatiota. Havainto johti tutkimukseen, jossa selvitettiin voisiko LKB1:n kasvun rajoitus välittyäkin epäsuorasti tukikudokseksi ajatelluista sileälihassoluista. Tätä tutkittiin kehittämällä poistogeeninen hiirimalli jossa Lkb1 on mutatoitunut vain sileälihassoluissa. Näille hiirille kehittyi polyyppeja, jotka ovat kaikin tavoin PJS-polyyppien kaltaisia. Lkb1:n menettäneiden solujen havaittiin tuottavan vähemmän transformoivaa kasvutekijä beetaa (TGFß), joka aiheutti solujen välisen viestinnän heikentymisen ja mahdollisesti viereisten epiteelisolujen liikakasvun. Vastaava häiriö havaittiin myös PJS-potilaiden polyypeissa, mikä viittaa siihen, että potilaillakin sileälihassolujen häiriö on polyyppien taustalla. Havainto suuntaa täten hoitokohteiden etsintää ja osoittaa että LKB1 toimii kasvunrajoittajana epätyypillisellä tavalla pitäen naapurisolujen kasvun kurissa.

VEGF-C/VEGFR-3 and PDGF-B/PDGFR-b pathways in embryonic lymphangiogenesis

The circulatory system comprises the blood vascular system and the lymphatic vascular system. These two systems function in parallel. Blood vessels form a closed system that delivers oxygen and nutrients to the tissues and removes waste products from the tissues, while lymphatic vessels are blind-ended tubes that collect extravasated fluid and cells from the tissues and return them back to blood circulation. Development of blood and lymphatic vascular systems occurs in series. Blood vessels are formed via vasculogenesis and angiogenesis whereas lymphatic vessels develop via lymphangiogenesis, after the blood vascular system is already functional. Members of the vascular endothelial growth factor (VEGF) family are regulators of both angiogenesis and lymphangiogenesis, while members of the platelet-derived growth factor (PDGF) family are major mitogens for pericytes and smooth muscle cells and regulate formation of blood vessels. Vascular endothelial growth factor C (VEGF-C) is the major lymphatic growth factor and signaling through its receptor vascular endothelial growth factor receptor 3 (VEGFR-3) is sufficient for lymphangiogenesis in adults. We studied the role of VEGF-C in embryonic lymphangiogenesis and showed that VEGF-C is absolutely required for the formation of lymph sacs from embryonic veins. VEGFR-3 is also required for normal development of the blood vascular system during embryogenesis, as Vegfr3 knockout mice die at mid-gestation due to failure in remodeling of the blood vessels. We showed that sufficient VEGFR-3 signaling in the embryo proper is required for embryonic angiogenesis and in a dosage-sensitive manner for embryonic lymphangiogenesis. Importantly, mice deficient in both VEGFR-3 ligands, Vegfc and Vegfd, developed a normal blood vasculature, suggesting VEGF-C- and VEGF-D- independent functions for VEGFR-3 in the early embryo. Platelet-derived growth factor B (PDGF-B) signals via PDGFR-b and regulates formation of blood vessels by recruiting pericytes and smooth muscle cells around nascent endothelial tubes. We showed that PDGF-B fails to induce lymphangiogenesis when overexpressed in adult mouse skin using adenoviral vectors. However, mouse embryos lacking Pdgfb showed abnormal lymphatic vessels, suggesting that PDGF-B plays a role in lymphatic vessel maturation and separation from blood vessels during embryogenesis. Lymphatic vessels play a key role in immune surveillance, fat absorption and maintenance of fluid homeostasis in the body. However, lymphatic vessels are also involved in various diseases, such as lymphedema and tumor metastasis. These studies elucidate the basic mechanisms of embryonic lymphangiogenesis and add to the knowledge of lymphedema and tumor metastasis treatments by giving novel insights into how lymphatic vessel growth could be induced (in lymphedema) or inhibited (in tumor metastasis).

New molecular strategies for prevention of fibroproliferative vascular disease : An experimental approach to restenosis

Vascular intimal hyperplasia is a major complication following angioplasty. The hallmark feature of this disorder is accumulation of dedifferentiated smooth muscle cells (SMCs) to the luminal side of the injured artery, cellular proliferation, migration, and synthesis of extracellular matrix. This finally results in intimal hyperplasia, which is currently considered an untreatable condition. According to current knowledge, a major part of neointimal cells derive from circulating precursor cells. This has outdated the traditional in vitro cell culture methods of studying neointimal cell migration and proliferation using cultured medial SMCs. Somatostatin and some of its analogs with different selectivity for the five somatostatin receptors (sst1 through sst5) have been shown to have vasculoprotective properties in animal studies. However, clinical trials using analogs selective for sst2/sst3/sst5 to prevent restenosis after percutaneous transluminal coronary angioplasty (PTCA) have failed to show any major benefits. Sirolimus is a cell cycle inhibitor that has been suggested to act synergistically with the protein-tyrosine kinase inhibitor imatinib to inhibit intimal hyperplasia in rat already at well-tolerated submaximal oral doses. The mechanisms behind this synergy and its long-term efficacy are not known. The aim of this study was to set up an ex vivo vascular explant culture model to measure neointimal cell activity without excluding the participation of circulating progenitor cells. Furthermore, two novel potential vasculoprotective treatment strategies were evaluated in detail in rat models of intimal hyperplasia and in the ex vivo explant model: sst1/sst4-selective somatostatin receptor analogs and combination treatment with sirolimus and imatinib. This study shows how whole vessel explants can be used to study the kinetics of neointimal cells and their progenitors, and to evaluate the anti-migratory and anti-proliferative properties of potential vasculoprotective compounds. It also shows how the influx of neointimal progenitor cells occurs already during the first days after vascular injury, how the contribution of cell migration is more important in the injury response than cell proliferation, and how the adventitia actively contribute in vascular repair. The vasculoprotective effect of somatostatin is mediated preferentially through sst4, and through inhibition of cell migration rather than of proliferation, which may explain why sst2/sst3/sst5-selective analogs have failed in clinical trials. Furthermore, a brief early oral treatment with the combination of sirolimus and imatinib at submaximal doses results in long-term synergistic suppression of intimal hyperplasia. The synergy is a result of inhibition of post-operative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury-site, and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation. In conclusion, the influx of progenitor cells already during the first days after injury and the high neointimal cell migratory activity underlines the importance of early therapeutic intervention with anti-migratory compounds to prevent neointimal hyperplasia. Sst4-selective analogs and the combination therapy with sirolimus and imatinib represent potential targets for the development of such vasculoprotective therapies.

Novel insights on functions of myotilin/palladin family members

Poikkijuovaisen luuranko- ja sydänlihaksen supistumisyksikkö, sarkomeeri, koostuu tarkoin järjestyneistä aktiini- ja myosiinisäikeistä. Rakenne eroaa muista solutyypeistä, joissa aktiinisäikeistö muovautuu jatkuvasti ja sen järjestyminen säätelee solun muotoa, solujakautumista, soluliikettä ja solunsisäisten organellien kuljetusta. Myotilin, palladin ja myopalladin kuuluvat proteiiniperheeseen, jonka yhteispiirteenä ovat immunoglobuliinin kaltaiset (Igl) domeenit. Proteiinit liittyvät aktiinitukirankaan ja niiden arvellaan toimivan solutukirangan rakenne-elementteinä ja säätelijöinä. Myotilinia ja myopalladinia ilmennetään poikkijuovaisessa lihaksessa. Sen sijaan palladinin eri silmukointimuotoja tavataan monissa kudostyypeissä kuten hermostossa, ja eri muodoilla saattaa olla solutyypistä riippuvia tehtäviä. Poikkijuovaisessa lihaksessa kaikki perheen jäsenet sijaitsevat aktiinisäikeitä yhdistävässä Z-levyssä ja ne sitovat Z-levyn rakenneproteiinia, -aktiniinia. Myotilingeenin pistemutaatiot johtavat periytyviin lihastauteihin, kun taas palladinin mutaatioiden on kuvattu liittyvän periytyvään haimasyöpään ja lisääntyneeseen sydäninfarktin riskiin. Tässä tutkimuksessa selvitettin myotilinin ja pallainin toimintaa. Kokeissa löydettiin uusia palladinin 90-92kDa alatyyppiin sitoutuvia proteiineja. Yksi niistä on aktiinidynamiikkaa säätelevä profilin. Profilinilla on kahdenlaisia tehtäviä; se edesauttaa aktiinisäikeiden muodostumista, mutta se voi myös eristää yksittäisiä aktiinimolekyylejä ja edistää säikeiden hajoamista. Solutasolla palladinin ja profilinin sijainti on yhtenevä runsaasti aktiinia sisältävillä solujen reuna-alueilla. Palladinin ja profilinin sidos on heikko ja hyvin dynaaminen, joka sopii palladinin tehtävään aktiinisäideiden muodostumisen koordinoijana. Toinen palladinin sitoutumiskumppani on aktiinisäikeitä yhteensitova -aktiniini. -Aktiniini liittää solutukirangan solukalvon proteiineihin ja ankkuroi solunsisäisiä viestintämolekyylejä. Sitoutumista välittävä alue on hyvin samankaltainen palladinissa ja myotilinissa. Luurankolihaksen liiallinen toistuva venytys muuttaa Z-levyjen rakennetta ja muotoa. Prosessin aikana syntyy uusia aktiinifilamenttejä sisältäviä tiivistymiä ja lopulta uusia sarkomeereja. Löydöstemme perusteella myotilinin uudelleenjärjestyminen noudattaa aktiinin muutoksia. Tämä viittaa siihen, että myotilin liittää yhteen uudismuodostuvia aktiinisäikeitä ja vakauttaa niitä. Myotilin saattaa myös ankkuroida viesti- tai rakennemolekyylejä, joiden tehtävänä on edesauttaa Z-levyjen uudismuodostusta. Tulostemme perusteella arvelemme, että myotilin toimii Z-levyjen rakenteen vakaajana ja aktiinisäikeiden säätelijänä. Palladinin puute johtaa sikiöaikaiseen kuolemaan hiirillä, mutta myotilinin puutoksella ei ole samanlaisia vaikutuksia. Tuotettujen myotilin poistogeenisten hiirten todetiin syntyvän ja kehittyvän normaalisti eikä niillä esiintynyt rakenteellisia tai toiminnallisia häiriöitä. Toisaalta aiemmissa kokeissa, joissa hiirille on siirretty ihmisen lihastautia aikaansaava myotilingeeni, nähdään samankaltaisia kuin sairailla ihmisillä. Näin ollen muuntunut myotilin näyttä olevan lihaksen toiminnalle haitallisempi kuin myotilinin puute. Myotilinin ja palladinin yhteisvaikutusta selvittääksemme risteytimme myotilin poistegeenisen hiiren ja hiirilinjan, joka ilmentää puutteellisesti palladinin 200 kDa muotoa. Puutteellisesti 200 kDa palladinia ilmentävien hiirten sydänlihaksessa todettiin vähäisiä hienorakenteen muutoksia, mutta risteytetyillä hiirillä tavattiin rakenteellisia ja toiminnallisia muutoksia myös luurankolihaksessa. Tulosten perusteella voidaan todeta, että palladinin 200 kDa muoto säätelee sydänlihassolujen rakennetta. Luurankolihaksessa sen sijaan myotilinilla ja palladinilla näyttäisi olevan päällekkäisiä tehtäviä.

CADASIL: molecular studies on the most common hereditary vascular dementing disorder

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common hereditary vascular dementia. CADASIL is a systemic disease of small and medium-sized arteries although the symptoms are almost exclusively neurological, including migraineous headache, recurrent ischemic episodes, cognitive impairment and, finally, subcortical dementia. CADASIL is caused by over 170 different mutations in the NOTCH3 gene, which encodes a receptor expressed in adults predominantly in the vascular smooth muscle cells. The function of NOTCH3 is not crucial for embryonic development but is needed after birth. NOTCH3 directs postnatal arterial maturation and helps to maintain arterial integrity. It is involved in regulation of vascular tone and in the wound healing of a vascular injury. In addition, NOTCH3 promotes cell survival by inducing expression of anti-apoptotic proteins. NOTCH3 is a membrane-spanning protein with a large extracellular domain (N3ECD) containing 34 epidermal growth factor-like (EGF) repeats and a smaller intracellular domain with six ankyrin repeats. All CADASIL mutations are located in the EGF repeats and the majority of the mutations cause gain or loss of one cysteine residue in one of these repeats leading to an odd number of cysteine residues, which in turn leads to misfolding of N3ECD. This misfolding most likely alters the maturation, targetting, degradation and/or function of the NOTCH3 receptor. CADASIL mutations do not seem to affect the canonical NOTCH3 signalling pathway. The main pathological findings are the accumulation of the NOTCH3 extracellular domain on degenerating vascular smooth muscle cells (VSMCs), accumulation of granular osmiophilic material (GOM) in the close vicinity of VSMCs as well as fibrosis and thickening of arterial walls. Narrowing of the arterial lumen and local thrombosis cause insufficient blood flow, mainly in small arteries of the cerebral white matter, resulting in tissue damage and lacunar infarcts. CADASIL is suspected in patients with a suggestive family history and clinical picture as well as characteristic white matter alterations in magnetic resonance imaging. A definitive verification of the diagnosis can be achieved by identifying a pathogenic mutation in the NOTCH3 gene or through the detection of GOM by electron microscopy. To understand the pathology underlying CADASIL, we have generated a unique set of cultured vascular smooth muscle cell (VSMC) lines from umbilical cord, placental, systemic and cerebral arteries of CADASIL patients and controls. Analyses of these VSMCs suggest that mutated NOTCH3 is misfolded, thus causing endoplasmic reticulum stress, activation of the unfolded protein response and increased production of reactive oxygen species. In addition, mutation in NOTCH3 causes alterations in actin cytoskeletal structures and protein expression, increased branching and abnormal node formation. These changes correlate with NOTCH3 expression levels within different VSMCs lines, suggesting that the phenotypic differences of SMCs may affect the vulnerability of the VSMCs and, therefore, the pathogenic impact of mutated NOTCH3 appears to vary in the arteries of different locations. Furthermore, we identified PDGFR- as an immediate downstream target gene of NOTCH3 signalling. Activation of NOTCH induces up-regulation of the PDGFR- expression in control VSMCs, whereas this up-regulation is impaired in CADASIL VSMCs and might thus serve as an alternative molecular mechanism that contributes to CADASIL pathology. In addition, we have established the congruence between NOTCH3 mutations and electron microscopic detection of GOM with a view to constructing a strategy for CADASIL diagnostics. In cases where the genetic analysis is not available or the mutation is difficult to identify, a skin biopsy is an easy-to-perform and highly reliable diagnostic method. Importantly, it is invaluable in setting guidelines concerning how far one should proceed with the genetic analyses.

GPRA and the asthma locus on chromosome 7p14-p15

The basis of this work was the identification of a genomic region on chromosome 7p14-p15 that strongly associated with asthma and high serum total immunoglobulin E in a Finnish founder population from Kainuu. Using a hierarchical genotyping approach the linkage region was narrowed down until an evolutionary collectively inherited 133-kb haplotype block was discovered. The results were confirmed in two independent data sets: Asthma families from Quebec and allergy families from North-Karelia. In all the three cohorts studied, single nucleotide polymorphisms tagging seven common gene variants (haplotypes) were identified. Over half of the asthma patients carried three evolutionary closely related susceptibility haplotypes as opposed to approximately one third of the healthy controls. The risk effects of the gene variants varied from 1.4 to 2.5. In the disease-associated region, there was one protein-coding gene named GPRA (G Protein-coupled Receptor for Asthma susceptibility also known as NPSR1) which displayed extensive alternative splicing. Only the two isoforms with distinct intracellular tail sequences, GPRA-A and -B, encoded a full-length G protein-coupled receptor with seven transmembrane regions. Using various techniques, we showed that GPRA is expressed in multiple mucosal surfaces including epithelial cells throughout the respiratory tract. GPRA-A has additional expression in respiratory smooth muscle cells. However, in bronchial biopsies with unknown haplotypes, GPRA-B was upregulated in airways of all patient samples in contrast to the lack of expression in controls. Further support for GPRA as a common mediator of inflammation was obtained from a mouse model of ovalbumin-induced inflammation, where metacholine-induced airway hyperresponsiveness correlated with elevated GPRA mRNA levels in the lung and increased GPRA immunostaining in pulmonary macrophages. A novel GPRA agonist, Neuropeptide S (NPS), stimulated phagocytosis of Esterichia coli bacteria in a mouse macrophage cell line indicating a role for GPRA in the removal of inhaled allergens. The suggested GPRA functions prompted us to study, whether GPRA haplotypes associate with respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) in infants sharing clinical symptoms with asthma. According to the results, near-term RDS and asthma may also share the same susceptibility and protective GPRA haplotypes. As in asthma, GPRA-B isoform expression was induced in bronchial smooth muscle cells in RDS and BPD suggesting a role for GPRA in bronchial hyperresponsiveness. In conclusion, the results of the present study suggest that the dysregulation of the GPRA/NPS pathway may not only be limited to the individuals carrying the risk variants of the gene but is also involved in the regulation of immune functions of asthma.

Lymphatic vascular maturation : Roles of FOXC2, NFATc1 and liprin ß1

The blood vascular system is a closed circulatory system, responsible for delivering oxygen and nutrients to the tissues. In contrast, the lymphatic vascular system is a blind-ended transport system that collects the extravasated tissue fluid from the capillary beds, and transports it back to the blood circulation. Failure in collecting or transporting the lymph, due to defects in the lymphatic vasculature, leads to accumulation of extra fluid in the tissues, and consequently to tissue swelling lymphedema. The two vascular systems function in concert. They are structurally related, but their development is regulated by separate, however overlapping, molecular mechanisms. During embryonic development, blood vessels are formed by vasculogenesis and angiogenesis, processes largely mediated by members of the vascular endothelial growth factor (VEGF) family and their tyrosine kinase receptors. The lymphatic vessels are formed after the cardiovascular system is already functional. This process, called lymphangiogenesis, is controlled by distinct members of the VEGF family, together with the transcription factors Prox1 and Sox18. After the primary formation of the vessels, the vasculature needs to mature and remodel into a functional network of hierarchically organized vessels: the blood vasculature into arteries, capillaries and veins; and the lymphatic vasculature into lymphatic capillaries, responsible for the uptake of the extravasated fluid from the tissues, and collecting vessels, responsible for the transport of the lymph back to the blood circulation. A major event in the maturation of the lymphatic vasculature is the formation of collecting lymphatic vessels. These vessels are characterized by the presence of intraluminal valves, preventing backflow of the lymph, and a sparse coverage of smooth muscle cells, which help in pumping the lymph forward. In our study, we have characterized the molecular and morphological events leading to formation of collecting lymphatic vessels. We found that this process is regulated cooperatively by the transcription factors Foxc2 and NFATc1. Mice lacking either Foxc2 or active NFATc1 fail to remodel the primary lymphatic plexus into functional lymphatic capillaries and collecting vessels. The resulting vessels lack valves, display abnormal expression of lymphatic molecules, and are hyperplastic. Moreover, the lymphatic capillaries show aberrant sprouting, and are abnormally covered with smooth muscle cells. In humans, mutations in FOXC2 lead to Lymphedema-Distichiasis (LD), a disabling disease characterized by swelling of the limbs due to insufficient lymphatic function. Our results from Foxc2 mutant mice and LD patients indicate that the underlying cause for lymphatic failure in LD is agenesis of collecting lymphatic valves and aberrant recruitment of periendothelial cells and basal lamina components to lymphatic capillaries. Furthermore, we show that liprin β1, a poorly characterized member of the liprin family of cytoplasmic proteins, is highly expressed in lymphatic endothelial cells in vivo, and is required for lymphatic vessel integrity. These data highlight the important role of FOXC2, NFATc1 and liprin β1 in the regulation of lymphatic development, specifically in the maturation and formation of the collecting lymphatic vessels. As damage to collecting vessels is a major cause of lymphatic dysfunction in humans, our results also suggest that FOXC2 and NFATc1 are potential targets for therapeutic intervention.

The mast cell as a regulator of atherosclerotic plaque stability

Atherosclerosis is an inflammatory disease progressing over years via the accumulation of cholesterol in arterial intima with subsequent formation of atherosclerotic plaques. The stability of a plaque is determined by the size of its cholesterol-rich necrotic lipid core and the thickness of the fibrous cap covering it. The strength and thickness of the cap are maintained by smooth muscle cells and the extracellular matrix produced by them. A plaque with a large lipid core and a thin cap is vulnerable to rupture that may lead to acute atherothrombotic events, such as myocardial infarction and stroke. In addition, endothelial erosion, possibly induced by apoptosis of endothelial cells, may lead to such clinical events. One of the major causes of plaque destabilization is inflammation induced by accumulated and modified lipoproteins, and exacerbated by local aberrant shear stress conditions. Macrophages, T-lymphocytes and mast cells infiltrate particularly into the plaque’s shoulder regions prone to atherothrombotic events, and they are present at the actual sites of plaque rupture and erosion. Two major mechanisms of plaque destabilization induced by inflammation are extracellular matrix remodeling and apoptosis. Mast cells are bone marrow-derived inflammatory cells that as progenitors upon chemotactic stimuli infiltrate the target tissues, such as the arterial wall, differentiate in the target tissues and mediate their effects via the release of various mediators, typically in a process called degranulation. The released preformed mast cell granules contain proteases such as tryptase, chymase and cathepsin G bound to heparin and chondroitin sulfate proteoglycans. In addition, various soluble mediators such as histamine and TNF-alpha are released. Mast cells also synthesize many mediators such as cytokines and lipid mediators upon activation. Mast cells are capable of increasing the level of LDL cholesterol in the arterial intima by increasing accumulation and retention of LDL and by decreasing removal of cholesterol by HDL in vitro. In addition, by secreting proinflammatory mediators and proteases, mast cells may induce plaque destabilization by inducing apoptosis of smooth muscle and endothelial cells. Also in vivo data from apoE-/- and ldlr-/- mice suggest a role for mast cells in the progression of atherosclerosis. Furthermore, mast cell-deficient mice have become powerful tools to study the effects of mast cells in vivo. In this study, evidence suggesting a role for mast cells in the regulation of plaque stability is presented. In a mouse model genetically susceptible to atherosclerosis, mast cell deficiency (ldlr-/-/KitW-sh/W-sh mice) was associated with a less atherogenic lipid profile, a decreased level of lipid accumulation in the aortic arterial wall and a decreased level of vascular inflammation as compared to mast-cell competent littermates. In vitro, mast cell chymase-induced smooth muscle cell apoptosis was mediated by inhibition of NF-kappaB activity, followed by downregulation of bcl-2, release of cytochrome c, and activation of caspase-8, -9 and -3. Mast cell-induced endothelial cell apoptosis was mediated by chymase and TNF-alpha, and involved chymase-mediated degradation of fibronectin and vitronectin, and inactivation of FAK- and Akt-mediated survival signaling. Subsequently, mast cells induced inhibition of NF-kappaB activity and activation of caspase-8 and -9. In addition, possible mast cell protease-mediated mechanisms of endothelial erosion may include degradation of fibronectin and VE-cadherin. Thus, the present results suggest a role for mast cells in destabilization of atherosclerotic plaques.

Immune mechanisms in atherosclerosis; Focus on the role of the complement system

Atherosclerosis is an inflammatory disease characterized by accumulation of lipids in the inner layer of the arterial wall. During atherogenesis, various structures that are recognized as non-self by the immune system, such as modified lipoproteins, are deposited in the arterial wall. Accordingly, atherosclerotic lesions and blood of humans and animals with atherosclerotic lesions show signs of activation of both innate and adaptive immune responses. Although immune attack is initially a self-protective reaction, which is meant to destroy or remove harmful agents, a chronic inflammatory state in the arterial wall accelerates atherosclerosis. Indeed, various modulations of the immune system of atherosclerosis-prone animals have provided us with convincing evidence that immunological mechanisms play an important role in the pathogenesis of atherosclerosis. This thesis focuses on the role of complement system, a player of the innate immunity, in atherosclerosis. Complement activation via any of the three different pathways (classical, alternative, lectin) proceeds as a self-amplifying cascade, which leads to the generation of opsonins, anaphylatoxins C3a and C5a, and terminal membrane-attack complex (MAC, C5b-9), all of which regulate the inflammatory response and act in concert to destroy their target structures. To prevent uncontrolled complement activation or its attack against normal host cells, complement needs to be under strict control by regulatory proteins. The complement system has been shown to be activated in atherosclerotic lesions, modified lipoproteins and immune complexes containing oxLDL, for instance, being its activators. First, we investigated the presence and role of complement regulators in human atherosclerotic lesions. We found that inhibitors of the classical and alternative pathways, C4b-binding protein and factor H, respectively, were present in atherosclerotic lesions, where they localized in the superficial proteoglycan-rich layer. In addition, both inhibitors were found to bind to arterial proteoglycans in vitro. Immunohistochemical stainings revealed that, in the superficial layer of the intima, complement activation had been limited to the C3 level, whereas in the deeper intimal layers, complement activation had proceeded to the terminal C5b-9 level. We were also able to show that arterial proteoglycans inhibit complement activation in vitro. These findings suggested to us that the proteoglycan-rich layer of the arterial intima contains matrix-bound complement inhibitors and forms a protective zone, in which complement activation is restricted to the C3 level. Thus, complement activation is regulated in atherosclerotic lesions, and the extracellular matrix is involved in this process. Next, we studied whether the receptors for the two complement derived effectors, anaphylatoxins C3a and C5a, are expressed in human coronary atherosclerotic lesions. Our results of immunohistochemistry and RT-PCR analysis showed that, in contrast to normal intima, C3aR and C5aR were highly expressed in atherosclerotic lesions. In atherosclerotic plaques, the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR, and a small fraction of them also expressed C3aR, mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. These results suggested that intimal cells can respond to and become activated by complement-derived anaphylatoxins. Finally, we wanted to learn, whether oxLDL-IgG immune complexes, activators of the classical complement pathway, could have direct cellular effects in atherogenesis. Thus, we tested whether oxLDL-IgG immune complexes affect the survival of human monocytes, the precursors of macrophages, which are the most abundant inflammatory cell type in atherosclerotic lesions. We found that OxLDL-IgG immune complexes, in addition to transforming monocytes into foam cells, promoted their survival by decreasing their spontaneous apoptosis. This effect was mediated by cross-linking Fc receptors with ensuing activation of Akt-dependent survival signaling. Our finding revealed a novel mechanism by which oxLDL-IgG immune complexes can directly affect the accumulation of monocyte-macrophages in human atherosclerotic lesions and thus play a role in atherogenesis.