Cardiovascular disease (CVD) is a major cause of adult morbidity and mortality in developed as well as in developing countries. In black population groups, stroke is more prominent than ischaemic heart disease. This may be attributed to a combination of risk factors seen in this population group inter alia raised haemostatic markers, which favour the development of stroke since it is well known that a disturbance in the haemostatic balance (a hypercoagulable and a hypofibrinolytic state) predisposes to CVD. It is generally accepted that childhood genetic, environmental and behavioural factors lay the groundwork for the manifestation of adult CVD. Therefore, one of the studies that form part of this dissertation was a cross-sectional study to determine whether haemostatic abnormalities are already present in black African adolescents and to determine whether high risk groups exist [in relation to the following haemostatic markers: fibrinogen, factor VIII (FVIII), plasminogen activator inhibitor type 1 activity (PAI-Iact), and thrombin anti-thrombin complex (TAT)] for the development of CVD later in life. The population subdivisions were made according to gender, body fat %, maturity status, height for age Z-score, and habitual PA levels. Since behavioural factors [diet, physical activity (PA), smoking and drinking habits] are controllable determinants, it could be possible to improve CVD risk to a certain degree. Therefore, the second study that forms part of this dissertation attempted to establish whether a PA programme will successfully reduce haemostatic variables in a subset of the study population used in the first study. The reader is referred to the abstracts at the beginning of each separate study manuscript (Chapters 3 and 4), for a description of the subjects, study design and methods used in each study. The results of the cross-sectional study showed that in African adolescents (a) gender independently contributed to the variability in PAI-Iact, but that the gender difference in fibrinogen and TAT could be explained by the significant differences in fat mass and PA levels observed between the genders; (b) fibrinogen was significantly higher in the stunted compared to the non-stunted children indicating that childhood chronic malnutrition may possibly predispose independently to CVD; (c) fitness influences TAT concentrations positively and that (d) no significant differences in FVIII could be found between any of the subdivisions. As these determinants seem to be modifiable through behavioural changes and optimal nutrition status through early life, it raises a sense of urgency to develop strategies for the prevention and treatment of these risk factors. The results of the intervention study showed that an 11-week outdoor PA intervention programme had no significant effect on the haemostatic markers of African adolescents, but the results of this study should be interpreted with caution since (a) seasonal variations could have clouded the effect of the PA intervention as baseline measurements were taken in the summer and end measurements in the winter; (b) attendance of the PA sessions does not necessarily implicate compliance to the exercises given; (c) baseline values seem to play a prominent role in the changes that could be expected during an intervention and, therefore, improvements in the haemostatic profile would most likely be more significant in individuals with raised baseline levels. Similar research on African children is warranted since studies investigating PA's effect on haemostatic variables remain a topic of debate and speculation and data on African population groups are scanty. / Thesis (M.Sc. (Nutrition))--North-West University, Potchefstroom Campus, 2007

Submitted by Irene Nascimento (irene.kessia@ufpe.br) on 2016-12-15T14:53:15Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) VersaoAtualizada2016 - Tese Georgge Gomes Oliveira - varsão para biblioteca UFPE.pdf: 3290385 bytes, checksum: 2ddc23c4486bcc121a9ea222566d1f09 (MD5) / Made available in DSpace on 2016-12-15T14:53:15Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) VersaoAtualizada2016 - Tese Georgge Gomes Oliveira - varsão para biblioteca UFPE.pdf: 3290385 bytes, checksum: 2ddc23c4486bcc121a9ea222566d1f09 (MD5) Previous issue date: 2015-04-27 / Ministério da Saúde do Brasil / Estudos recentes mostram que a síndrome metabólica (SM) é freqüente nas pessoas vivendo com HIV/AIDS (PLWHA). A importância na identificação da SM baseia-se no aumento do risco em cinco vezes de desenvolver diabetes mellitus tipo 2 (DM2) e em duas vezes de apresentar doença cardiovascular (DCV) trombóticas, embora os fatores de hipercoagulabilidade não estejam incluídos nos critérios de definição da síndrome. A SM é caracterizada pela presença concomitante de fatores reconhecidamente aterogênicos em um mesmo indivíduo. A freqüência de DCV em PLWHA vem aumentando ao longo dos anos. O PAI-1 é uma proteína importante na cascata de fibrinólise e seu aumento está associado ao estado de hipercoagulabilidade. Sua regulação depende de fatores genéticos, dentre eles, destaca-se o polimorfismo 4G5G do gene SERPINE1. A participação de substâncias protrombóticas na doença cardiovascular é conhecida em pessoas sem HIV, porém menos elucidada em PLWHA. Diante disto o objetivo deste trabalho foi determinar a freqüência do polimorfismo 4G5G em pessoas que vivem com HIV e verificar se o polimorfismo tem associação com a expressão do PAI-1 plasmático, com SM e com risco cardiovascular (RCV) estimado pelo escore de Framingham. Também objetivamos verificar associação dos níveis de PAI-1 com RDC e com SM. Para tanto foi desenvolvido estudo transversal para determinação da freqüência do polimorfismo 4G5G do PAI-1 e estudo tipo caso-controle para verificar associações entre polimorfismos com níveis plasmáticos de PAI-1 com SM e depois com RCV. Também foram testadas associações com fatores de risco tradicionais. Para primeiro estudo a amostra foi 185 pessoas sorteadas de um grupo de 2074 participantes da Cohort AIDS-PE Study Group. A prevalência de heterozigose foi de 86,8% e homozigose para 4G4G de 4,4%. A média de idade foi de 40,5 (DP ± 9,9 anos). A mediana de PAI-1 ativado foi de 13,6 ng/mL (IQ: 10,8-17,5). A freqüência de SM foi de 37,9% e de dislipidemia de 82,4%. Não encontramos associação do polimorfismo com os níveis plasmáticos de PAI-1, nem com SM. Para o segundo estudo houve perda de 23, restando 162 pessoas das quais 72,8% era do sexo feminino e a média de idade foi de 40 anos. A freqüência de RDCV estimado > 10% foi de 10,5%. O alelo 4G esteve presente em 91,0% das pessoas (genótipos 4G4G e 4G5G). Não houve associação entre polimorfismo e RDCV estimado > 10% (OR=0,6; IC95% 0,1 – 3,7), nem diferença dos níveis de PAI-1 em relação ao RDCV estimado (RCV>10% 14,6 ng/ml x RDCV < 10% 14,1 ng/ml; ρ=0,9). Hipercolesterolemia foi associada com genótipo 5G5G do polimorfismo (OR: 3,3; IC95%: 1,25 – 10) e com níveis plasmáticos mais elevados do PAI-1 (colesterol não HDL (CNHDL) > 130 mg/dl = 15,6 ng/ml versus CNHDL < 130 ng/ml = 13,8 ng/ml; ρ=0,04). Nesse estudo, encontramos alta prevalência do heterozigose para o polimorfismo 4G5G em pessoas vivendo com HIV/AIDS, no nordeste do Brasil. Entretanto, não encontramos associação entre o polimorfismo estudado com níveis plasmáticos de PAI-1 nem com SM. Também não verificamos associação do polimorfismo 4G5G do PAI-1 nem dos níveis plasmáticos de PAI-1 com RCV>10% pelo escore de Framingham, mas houve com hipercolesterolemia. / Recent studies show that the metabolic syndrome (MS) is common in people living with HIV / AIDS (PLWHA). The importance of identifying MS is based on an increased risk of developing fivefold type 2 diabetes mellitus (T2DM) and twice presenting cardiovascular disease (CVD) thrombotic, although hypercoagulability factors are not included in the definition of criteria syndrome. MS is characterized by the concomitant presence of known atherogenic factors in the same individual. The frequency of CVD in PLWHA has increased over the years. The PAI-1 is an important protein in the fibrinolytic cascade and its increase is associated with the hypercoagulable state. Its regulation depends on genetic factors, among them stands out the 4G5G polymorphism SERPINE1 gene. The participation of prothrombotic substances in cardiovascular disease is known in people without HIV, but less elucidated in PLWHA. In view of this the objective of this study was to determine the frequency of 4G5G polymorphism in people living with HIV and verify that polymorphism is associated with the expression of PAI-1 plasma with MS and cardiovascular risk (RCVD) estimated by the Framingham score . We aim to also assess the association of PAI- 1 levels with CVD and with MS. For this cross-sectional study was developed to determine the frequency of 4G5G polymorphism of PAI- 1 and case-control study to examine associations between polymorphisms and plasma levels of PAI- 1 with SM and then RCVD. Associations were also tested with traditional risk factors. For the first study sample was randomly selected 185 people of a group of participants 2074 of AIDS-PE Cohort Study Group. The prevalence of heterozygosity was 86.8% and homozygous for 4G4G 4.4%. The average age was 40.5 (SD ± 9.9 years). The PAI-1 activated median was 13.6 ng/mL (CI: 10.8 to 17.5). The frequency of MS was 37.9% and 82.4% dyslipidemia. We did not find polymorphism association with plasma levels of PAI-1 or with SM. For the second study, there was loss of 23, leaving 162 people of which 72.8% were female and the average age was 40 years. The frequency of RDCV estimated> 10% was 10.5%. The 4G allele was present in 91.0% of people (4G4G and 4G5G genotypes). There was no association between polymorphism and RDCV estimated> 10% (OR = 0.6; 95% CI 0.1 to 3.7), or difference of PAI-1 levels relative to estimated RDCV (RCV> 10% 14.6 ng / ml x RDCV <10% 14.1 ng/ml; ρ = 0.9). Hypercholesterolemia was associated with 5G5G genotype polymorphism (OR: 3.3; 95% CI: 1.25 to 10) and higher plasma levels of PAI-1 (non-HDL cholesterol (CNHDL)> 130 mg / dl = 15.6 ng/ml CNHDL versus <130 ng/ml = 13.8 ng/ ml; ρ = 0.04). In this study, we found a high prevalence of heterozygous for the polymorphism 4G5G in people living with HIV/AIDS, in northeastern Brazil. However, we found no association between the polymorphism studied with plasma levels of PAI-1 or with SM. Nor do we find polymorphism association 4G5G of PAI-1 or plasma levels of PAI-1 with RCV> 10% by Framingham score, but happened to hypercholesterolemia.

O endotélio reveste a parede vascular e possui função essencial na manutenção da homeostase. A célula endotelial é capaz de perceber estímulos extracelulares, como fatores químicos e mecânicos, transmitir a informação para dentro da célula e regular sua função e fenótipo. Neste sentido, investigamos os mecanismos moleculares associados as células endoteliais em dois contextos importantes de intervenções vasculares 1) nos stents farmacológicos, onde a rapamicina exerce funções antiproliferativas e pró-trombogênicas, e 2) na revascularização cardíaca por ponte de safena, onde o alto estiramento mecânico exerce grande impacto no remodelamento vascular e no fenótipo da célula endotelial. A rapamicina pertence à classe de drogas limus, bastante utilizadas nos stents farmacológicos usados no procedimento de desobstrução vascular. Além de sua função antiproliferativa, exploramos os efeitos deletérios associados a pró-trombogênese. Os dados demonstraram que a rapamicina ativa o receptor de TGF independentemente de seu ligante TGFbeta, promovendo aumento na expressão da PAI-1 (pró-trombogênica), alteração no fenótipo endotelial (Transição endotélio-mesenquimal - EndMT) e na formação de fibras de estresse. Os efeitos observados são dependentes da ativação de Smad2 e independentes da via clássica antiproliferativa por mTOR. Experimentos in vivo mostraram que o tratamento com inibidor do receptor de TGF diminui os efeitos pró-trombogênicos e a expressão de PAI-1 induzidos pela rapamicina em artérias carótidas de camundongos. A ponte de safena é um procedimento bastante utilizado na cirurgia de revascularização cardíaca e a arterialização do segmento venoso submetido ao estresse hemodinâmico arterial resulta em remodelamento vascular, que influencia o sucesso do procedimento. Nossos dados demonstram que a célula endotelial humana de veia safena humana (hSVEC), susceptível as modificações do tipo EndMT induzido quimicamente (estímulo pró-fibrótico e pró-inflamatório), não expressou o mesmo comportamento em resposta ao aumento de estiramento mecânico que ocorre durante a arterialização venosa. Entretanto, detectamos uma pronunciada redução dos filamentos de actina, modulação no padrão de ativação da cofilina e na proporção de actina glomerular (G-actina) entre citoplasma e núcleo, com redução da biodisponibilidade de NO. De modo interessante, demonstramos que a redução no filamento de actina é específica para a célula endotelial venosa, não sendo observado em células endoteliais de origem arterial de aorta e coronária. Em conjunto, os dados mostram que 1) efeitos pró-trombogênicos associados a rapamicina são mediados por ativação do receptor de TGF independente do seu ligante e da atividade antiproliferativa da droga e 2) a adaptação da célula endotelial venosa ao estiramento mecânico envolve modulação da síntese/degradação de filamentos de actina e redução na biodisponibilidade de NO. Estes novos elementos sobre o mecanismo de transdução de estímulos químicos e físicos pelo endotélio poderão ser explorados terapeuticamente para modular a plasticidade endotelial em disfunções cardiovasculares / Endothelium is the inner layer in vascular wall and displays an essential role in the maintenance of cardiovascular homeostasis. Endothelial cell senses the extracellular stimuli, such as chemical and mechanical factors, transduce and process these signals to regulate cell function and phenotype. Here, we investigated molecular underpinning of the endothelial cells under two important scenarios: 1) in drug-eluting stents, where rapamycin exerts antiproliferative and undesirable prothrombogenic functions, and 2) in vein graft bypass surgery, where increased stretch modulates vascular remodeling and endothelial cell phenotype. Rapamycin belongs to the class of limus drugs and is widely used in drug eluting stents (DES) to vascular restenosis. In addition to its antiproliferative function, we explore the deleterious effects associated with prothrombogenesis. Our data demonstrated that rapamycin activates TGF receptor independent of its ligand TGFbeta, in concert with promotion of PAI-1 expression (prothrombogenic), changes in endothelial phenotype (Endothelial to Mesenchymal Transition - EndMT) and stress fibers induction. These effects are Smad2 dependent and independent of the classical antiproliferative mTOR pathway of rapamycin. Our in vivo experiments showed that TGF receptor inhibitor treatment decreases prothrombogenic effects and PAI-1 expression induced by rapamycin in mice carotid arteries. Saphenous vein is widely used in coronary artery bypass surgery (CABG) and the vein arterialization remodeling in response to the increased stress influences graft patency. Our data demonstrated that human saphenous vein endothelial cell (hSVEC) is susceptible to chemically induced endothelial-to-mesenchymal transition (EndMT) by pro-fibrotic and pro-inflammatory stimuli. On the other hand, physical stimulus associated with high stretch failed to induce EndMT. However, we detected a pronounced decrease of actin filaments, modulation of the cofilin activation, changes in the proportion of glomerular actin (G-actin) between cytoplasm and nucleus, and reduction of NO bioavailability. Interestingly, the reduction of actin fibers by high stretch is specific to venous endothelial cell since arterial endothelial cells from aorta, and coronary artery failed to display the response. Altogether, our data show that 1) the thrombogenic effects of rapamycin are mediated by TGF receptor activation independent of its ligand and independent of the antiproliferative pathway of the drug, and 2) the adaptation of venous endothelial cell to mechanical stretch involves synthesis/degradation of actin filaments and reduced NO bioavailability. These new elements on signal transduction of endothelial cells in response to chemical and physical stimuli may be therapeutically explored to modulate endothelial plasticity in cardiovascular disorders

Le facteur 4 plaquettaire (PF4/CXCL4) est un tétramère constitué de quatre sous-unités identiques de 7,8 kDa qui est libéré en grande quantité par les plaquettes lors de l’hémostase primaire (ensemble des phénomènes permettant un colmatage initial d’une lésion vasculaire). L’étude de la formation d’un caillot de fibrine en présence de PF4 montre une augmentation de la turbidité finale du caillot : le PF4 modifie le réseau formé. Etant donné que la plupart des acteurs de la fibrinolyse se lie au caillot de fibrine et que le PF4 modifie sa structure, nous avons pensé qu’il serait intéressant de rechercher si le PF4 influençait aussi la fibrinolyse. La lyse d'un caillot est effectuée par la plasmine issue de l'activation du plasminogène par son activateur d’origine tissulaire (t-PA) en présence d’un cofacteur qui n'est autre que la fibrine. Nous avons étudié la lyse de caillots de plasma, obtenus par activation de la cascade de la coagulation, en condition statique et à l'aide d'un modèle de thrombose artérielle (système Chandler loop). Dans les deux cas, une diminution du temps de demi-lyse a été observée en présence de PF4. Cependant, la lyse de caillots préparés par simple ajout de thrombine sur du fibrinogène ne permet pas de retrouver cet effet du PF4. Ceci suggère que l’influence du PF4 sur la structure du caillot n’est pas à l’origine de l’effet sur sa lyse et que le PF4 n’influence pas (ou très peu) l'activation du plasminogène, ainsi que l'activité de la plasmine résultante. Cette hypothèse a été confirmée par l’étude de l’activité amydolytique du t-PA et de la plasmine (quelle soit ajoutée ou générée). En système purifié, les inhibiteurs plasmatiques de la fibrinolyse sont absents. Les deux principaux sont l'inhibiteur de l'activateur du plasminogène de type 1 (PAI-1) et l’α2-antiplasmine (α2-AP). La lyse de caillots préparés à partir de plasma déficient en α2-AP montre une diminution du temps de demi-lyse en présence de PF4 (comme pour le plasma normal), alors qu’avec le plasma dépourvu de PAI-1 le temps de demi-lyse n'est plus influencé. De plus, l’ajout de PAI-1 dans le système purifié entraine une diminution du temps de demi-lyse en présence de PF4. Ceci suggère que le PF4 prévient directement ou indirectement l'inhibition du t-PA par PAI-1. L’étude de la cinétique d'inhibition de l’activité amidolytique du t-PA par le PAI-1, la détermination de la stœchiométrie de cette inhibition, et l’analyse de ces cinétiques par immuno-empreinte montrent que le PF4 est un modulateur de la fibrinolyse qui agit en retardant la formation d'un complexe initial entre le t-PA et le PAI-1. Cette nouvelle fonction du PF4 est cohérente, et vient en complément de celle décrite récemment d’inhibiteur de l'activation du TAFI. / Platelet factor 4 (PF4/CXCL4) is a tetramer constituted of four identical 7,8 kDa subunits released in large quantities by platelets during primary heamostasis (allowing initial clogging of a vascular injury). Study of fibrin clot formation in the presence of PF4 shows an increase of the final clot turbidity: PF4 modifies the formed network. Given that most fibrinolysis actors are bound to the fibrin clot and that PF4 modifies its structure we thought it would be interesting to investigate if PF4 also influences fibrinolysis. Clot lysis is performed by plasmin originating from activation of its precursor by tissue plasminogen activator (t-PA) with fibrin itself as cofactor of the reaction. We have studied lysis of plasma clots formed by activation of the coagulation cascade in static condition and in a Chandler loop model mimicking arterial thrombosis. Half-times of lysis decreased in the presence of PF4 in both systems. However, PF4 had no longer detectable influence on the half-time of lysis with clots formed by direct addition of thrombin on purified fibrinogen. Observation suggested that the observed decrease of the half-time of lysis induced by PF4 did not originate from its influence on fibrin clot formation and that PF4 had little effect if any on plasminogen activation or plasmin activity. We confirmed this hypothesis by comparing amydolytic activities of t-PA and plasmin (added or generated through plasminogen activation). In purified system, fibrinolysis inhibitors are absent. The two main inhibitors are plasminogen activator inhibitor-1 (PAI-1) and α2-antiplasmin (α2-AP). Lysis of clots obtained from α2-AP deficient plasma showed a decrease of the half-time of lysis in the presence of PF4 (as in normal plasma), whereas in PAI-1 deficient plasma half-time of lysis was unchanged. Moreover if PAI-1 was added to the purified system, half-time of lysis decreased in the presence of PF4. Observations therefore suggested that PF4 prevented directly or indirectly t-PA inhibition by PAI-1. Kinetics of the amidolytic activity of t-PA inhibition by PAI-1 in the presence or not of PF4, determination of its stoichiometry and Western blot analysis of these inhibition kinetics revealed that PF4 is a fibrinolysis modulator which delays formation of the initial (Michaelis) complex between t-PA and PAI-1. This new feature of PF4 is consistent and complementary with its recently described role as a modulator of TAFI activation.

<p>Tromboza danas, u većini razvijenih zemalja, predstavlja vodeći uzrok obolevanja i umiranja. Poslednjih godina veoma aktuelna su istraživanja venskog tromboembolizma, obzirom da je incidenca ovog oboljenja 2/1000 osoba godi&scaron;nje, a njegov razvoj posledica udruženog delovanja vi&scaron;e genetskih i stečenih faktora rizika. &Scaron;to preciznije prepoznavanje i sagledavanje &scaron;to većeg broja ovih faktora osnovni je cilj u borbi, kako protiv prve epizode venske tromboze, tako i protiv recidiva ove bolesti. Brojni faktori rizika već su prepoznati kao sastavne karike patofiziolo&scaron;kog lanca venskog trombotskog procesa, ali je evidentno da otkrića mnogih od njih tek predstoje. Među najaktulenijim istraživanjima na ovom polju nalazi se i ispitivanje uloge poremećaja fibrinoliznog mehanizma u venskoj tromboembolijskoj bolesti. Iako su već pruženi dokazi da suprimirana fibrinolizna aktivnost povećava rizik od nastanka ovog oboljenja, jo&scaron; uvek postoje brojna otvorena pitanja, koja se pre svega odnose na ulogu pojedinačnih činilaca fibrinoliznog mehanizma u venskoj trombozi, kao i na globalnu ulogu fibrinoliznog mehanizma u različitim tipovima i lokalizacijama venske trombotske bolesti. Pored toga, ispitivanje uticaja pojedinih genskih mutacija na pojadinačne činioce fibrinoliznog mehanizma, njegovu globalnu funkcionalnost i posredno na rizik za nastanak venske tromboze, takođe zaokuplja pažnju stručne javnosti, obzirom na nekonzistentnost rezultata dobijenih studijama koje se bave ovom problematikom. Cilj ovoga istraživanja je ispitivanje kako globalne funkcionalnosti fibrinoliznog mehanizma, tako i njegovih pojedinačnih činilaca, kod bolesnika sa različitim tipovima i lokalizacijama venske tromboze i poređenje ovih parametara sa njihovim vrednostima u zdravoj populaciji. Pored toga, cilj istraživanja je i ispitivanje zastupljenosti 4G/5G PAI-1 polimorfizma kod bolesnika sa venskom trombozom u poređenju sa zdravim osobama. Ispitivanu grupu je sačinjavalo 100 bolesnika koji su doživeli trombozu dubokih vena a kontrolnu grupu je činilo 100 zdravih ispitanika, koji nikada nisu imali trombozni incident. Iz ispitivanja su isključene: osobe sa prethodno dokazanim poremećajem hemostaznog mehanizma, osobe koje uzimaju lekove za koje se zna da mogu imati uticaja na hemostazni mehanizam, osobe koje su imale akutnu bolest u momentu uzorkovanja krvi ili 6 nedelja pre toga, osobe sa malignitetom, trudnice, osobe sa težim du&scaron;evnim bolestima, bolestima jetre i bubrega, autoimunim bolestima, ispitanici koji su odbili da potpi&scaron;u pristanak informisanog ispitanika. Kao test za procenu globalne funkcionalnosti fibrinoliznog mehanizma kori&scaron;teno je euglobulinsko vreme lize koaguluma, dok su od pojedinačnih činilaca određivani: tkivni aktivator plazminogena (t-PA) i trombinom aktivi&scaron;ući fibrinolizni inhibitor (TAFI) - ELISA metodom, kao i inhibitor aktivatora plazminogena-1 (PAI-1) - metodom hromogenog substrata. Genetskim ispitivanjem je utvrđivano prisustvo PAI-1 4G/5G genskog polimorfizma. Prema rezultatima istraživanja kod 56% bolesnika bila je prisutna spontana venska tromboza, dok je 44% njih imalo trombozu provociranu jednim od priznatih faktora rizika. U odnosu na lokalizaciju venskog tromboznog procesa proksimalna venska tromboza bila je prisutna kod 63% bolesnika, izolovana distalna venska tromboza kod 29% bolesnika, a atipično lokalizovana venska tromboza kod 8% bolesnika. Posmatrajući zastupljenost pojedinih faktora rizika uočili smo da je značajno vi&scaron;i procenat osoba sa hipertenzijom bio prisutan u grupi bolesnika sa primarnom trombozom dubokih vena u odnosu na grupu bolesnika sa provociranom trombozom dubokih vena (61% vs.16%; p=0.000). &Scaron;to se funkcionalnosti fibrinoliznog mehanizma tiče, prema na&scaron;im rezultatima bolesnici koji su doživeli trombozu dubokih vena imaju značajno duže vreme lize koaguluma, odnosno suprimiranu funkcionalnost fibrinolize u poređenju sa zdravim kontrolama (204.34&plusmn;51.24 vs. 185.62&plusmn;42.30; p=0.011), a kada posmatramo podgrupe bolesnika u odnosu na lokalizaciju i vrstu venske tromboze uočavamo da podgrupa bolesnika sa izolovanom distalnom venskom trombozom ima značajno duže euglobulinsko vreme lize koaguluma u odnosu na kontrolnu grupu (218.32&plusmn;41.12 vs.185.62&plusmn;42.30: p=0.001), kao i bolesnici koji su imali provociranu vensku trombozu u poređenju sa kontrolama (208.18&plusmn;48.53 vs. 185.62&plusmn;42.30; p=0.018). Ispitivanjem pojedinačnih komponenti fibrinoliznog mehanizma do&scaron;li smo do rezultata da bolesnici koji su doživeli venski trombozni incident imaju značajno vi&scaron;e koncentracije TAFI u poređenju sa osobama koje nikada nisu imale vensku trombozu (19.70 ng/ml &plusmn; 5.17 vs.17.13 ng/ml &plusmn; 4.25; p=0.001). Poređenjem bolesnika sa provociranom trombozom dubokih vena i kontrolnih ispitanika uočili smo da bolesnici iz ove podgrupe imaju značajno vi&scaron;e vrednosti plazminogena u poređenju sa zdravim osobama (127.14 % &plusmn; 27.73 vs.117.09 % &plusmn; 24.49; p= 0.044), kao i značajno vi&scaron;e koncentracije t-PA (20.02 ng/ml &plusmn; 11.07 vs. 16.78 ng/ml &plusmn; 8.08; p=0.042). &Scaron;to se tiče TAFI, bolesnici sa distalnom trombozom dubokih vena u poređenju sa kontrolama (20.72 ng/ml &plusmn; 4.96 vs.17.13 ng/ml &plusmn; 4.25; p=0.001), kao i bolesnici sa proksimalnom trombozom dubokih vena u poređenju sa kontrolama (19.37 ng/ml &plusmn; 5.33 vs.17.13 ng/ml &plusmn; 4.25; p=0.013) imaju značajno vi&scaron;e koncentracije TAFI. Koncentracija ovog inhibitora fibrinoliznog procesa značajno je veća i kod bolesnika sa provociranom trombozom dubokih vena u poređenju s zdravim osobama (19.93 ng/ml &plusmn; 3.97 vs.17.13 ng/ml &plusmn; 4.25; p=0.000), kao i kod bolesnika sa primarnom trombozom dubokih vena u poređenju sa zdravim ispitanicima (19.53 ng/ml &plusmn; 5.97 vs.17.13 ng/ml &plusmn; 4.25; p=0.023). &Scaron;to se genetskih analiza tiče, u okviru grupe bolesnika imali smo 25% homozigotnih i 58% heterozigotnih nosilaca mutacije gena za PAI-1, dok 17% bolesnika nije imalo pomenutu gensku mutaciju. U okviru kontrolne grupe pak, bilo je 30% homozigotnih i 56% heterozigotnih nosilaca mutacije a 14% ispitanika nije imalo mutaciju. Nije uočena značajna razlika u zastupljenosti 4G/4G genotipa između bolesnika sa različitim lokalizacijama venskog trombotskog procesa (distalna DVT 29% vs. proksimalna DVT 21% vs. DVT retke lokalizacije 12%; p=0.501), kao ni u zastupljenosti ovoga genotipa kod provocirane i spontane tromboze dubokih vena (27% vs. 23%; p=0.642), niti kod izolovane tromboze dubokih vena u poređenju sa plućnom tromboembolijom (25% vs. 33%; p=0.735). Procena rizika za nastanak venske tromboze u odnosu na postojanje poremećaja globalne funkcionalnosti fibrinoliznog mehanizma, u odnosu na patolo&scaron;ke koncentracije pojedinih komponenti fibrinoliznog mehanizma, kao i u odnosu na postojanje 4G/4G mutacije u genu za PAI-1, pokazala je da suprimirana funkcionalnost fibrinoliznog mehanizma trostruko povećava rizik za nastanak tromboze dubokih vena (OR 3.02; CI 1.26-7.22), povi&scaron;en nivo PAI-1 nema uticaja na rizik od nastanka tromboze dubokih vena, na &scaron;ta ukazuje OR od 0.86 sa CI 0.59-1.25, povi&scaron;en nivo t-PA antigena ne utiče na rizik od nastanka tromboze dubokih vena (OR 1.53; CI 0.79-2.94), ali povi&scaron;ena koncentracija TAFI vi&scaron;e od dvostruko povećava ovaj rizik (OR 2.25; CI 1.16-4.35). Prema na&scaron;im rezultatima PAI-1 4G/4G genotip nema uticaja na rizik od nastanaka venske tromboze, &scaron;to potvrđuje OR koji iznosi 0.57 (0.27-1.20). Na osnovu dobijenih rezultata zaključujemo da bolesnici sa trombozom dubokih vena imaju suprimiranu funkcionalnost fibrinoliznog mehanizma u poređenju sa zdravim osobama, da je nivo t-PA antigena, kao i plazminogena značajno vi&scaron;i kod bolesnika sa provociranom venskom trombozom nego kod zdravih osoba, da nema razlike u koncentraciji PAI-1 između bolesnika sa venskom trombozom i zdravih osoba, ali da bolesnici sa trombozom dubokih vena, bez obzira na njenu lokalizaciju ili vrstu imaju značajno vi&scaron;e nivoe TAFI u poređenju sa zdravim ispitanicima. Pored toga možemo zaključiti da ne postoji razlika u zastupljenosti 4G/5G polimorfizma između bolesnika sa venskom trombozom i zdravih ispitanika. Konačno, možemo reći da na osnovu na&scaron;ih rezultata možemo zaključiti da suprimirana funkcionalnost fibrinoliznog mehanizma trostruko povećava rizik od nastanka tromboze dubokih vena, a povi&scaron;en nivo TAFI-a dvostruko povećava ovaj rizik, dok 4G/5G PAI-1 polimorfizam nema uticaja na rizik za nastanak venskog tromboembolizma.</p> / <p>Thrombosis is nowadays leading cause of morbidity and mortality worldwide. Lately, studies dealing with venous thromboembolism are very actual, since incidence of this disease is 2/1000 persons per year and its development is consequence of joint action of many different inherited and acquired risk factors. Precise recognition and understanding as many of those factors as possible represents imperative in fight against the first episode of venous thrombosis, and also against the recurrence of the disease. Numerous risk factors have been already recognized as constituent links of pathophysiological chain of venous thrombotic process, but it is also clear that the discovery of many of them are yet to come. Investigations of the role of fibrinolytic mechanism disorders in venous thrombosis are topical in the field. Although, we have some evidences that suppressed fibrinolytic activity increases the risk of this disease, still there are many open issues, especially those dealing with the role of individual factors of fibrinolytic mechanism in venous thrombosis, and with the role of global fibrinolytic function in different types and localizations of venous thrombotic disease. Further, investigation of the effects of gene mutations on individual fibrinolytic mechanism components, its global functionality and indirectly to the risk of venous thrombosis, also attracts the attention of experts, given the inconsistency of results obtained from studies dealing with this issue. The aim of this study was to evaluate fibrinolytic mechanism global functionality, as well as functionality of its integral individual components in patients with different venous thrombosis types and localizations, and to compare them with those of the healthy persons. In addition, the aim was to evaluate presence of 4G/5G PAI-1 polymorphism in patients with venous thrombosis compared with healthy subjects. The case group consisted of 100 patients with deep vein thrombosis and the control group consisted of 100 healthy subjects who had never had thrombotic incident. Exclusion criteria were: documented haemostatic disease, taking drugs proven to affect fibrinolytic function, acute illness within 6 weeks before blood sampling, malignancy, pregnancy, severe mental illness, kidney or liver diseases, autoimmune diseases, examinee refusal to sign the informed consent. We used euglobulin cloth lysis time test as test for global fibrinolytic mechanism function estimation, and also determined: t-PA and TAFI concentrations using ELISA method and PAI-1 concentrations using chromogenic substrate method. The presence of PAI-1 4G/5G gene polymorphism was determined by genetic testing. According to results 56% of patients had unprovoked and 44% had provoked venous thrombosis. Proximal venous thrombosis was present in 63% of cases, distal venous thrombosis in 29% of cases and atypical venous thrombosis in 8% of them. Significantly higher frequency of hypertension was present in patients with primary deep vein thrombosis than in the group of patients with provoked deep vein thrombosis (61% vs. 16%, p = 0.000). Patients who have experienced deep vein thrombosis had a significantly longer clot lysis time, and suppressed fibrinolysis function compared with healthy controls (204.34 &plusmn; 51.24 vs. 185.62 &plusmn; 42.30, p = 0.011). Also, this parameter was significantly longer in patients with isolated distal deep vein thrombosis compared with healthy controls (218.32&plusmn;41.12 vs. 185.62&plusmn;42.30: p=0.001), such as in patients with provoked venous thrombosis compared with controls (208.18&plusmn;48.53 vs. 185.62&plusmn;42.30; p=0.018). Patients with venous thrombosis had significantly higher TAFI concentrations in comparison with healthy volunteers (19.70 ng/ml &plusmn; 5.17 vs. 17.13 ng/ml &plusmn; 4.25; p=0.001). Patients with provoked venous thrombosis had significantly higher concentrations of plasminogen (127.14 % &plusmn; 27.73 vs. 117.09 % &plusmn; 24.49; p= 0.044) and t-PA (20.02 ng/ml &plusmn; 11.07 vs. 16.78 ng/ml &plusmn; 8.08; p=0.042), in comparison with controls. Regarding TAFI, we noticed that patients with isolated distal deep vein thrombosis have higher values of this parameter compered with healthy people (20.72 ng/ml &plusmn; 4.96 vs. 17.13 ng/ml &plusmn; 4.25; p=0.001), such as patients with proximal deep vein thrombosis (19.37 ng/ml &plusmn; 5.33 vs. 17.13 ng/ml &plusmn; 4.25; p=0.013). The same was obtained when compared patients with provoked venous thrombosis and controls (19.93 ng/ml &plusmn; 3.97 vs. 17.13 ng/ml &plusmn; 4.25; p=0.000), and patients with unprovoked venous thrombosis and controls (19.53 ng/ml &plusmn; 5.97 vs. 17.13 ng/ml &plusmn; 4.25; p=0.023). As far as genetic analysis, in the group of patients we had 25% homozygous and 58% heterozygous carriers of PAI-1 gene mutation, whereas 17% of patients did&#39;t have this mutation. In controls, we had 30% homozygous and 56% heterozygous carriers of mutation and 14% of those without mutation. There was no significant difference in the frequency of 4G/4G genotype between patients with different localization of venous thrombotic process (distal DVT 29% vs. proximal DVT 21% vs. rare localization DVT 12%, p = 0.501), as well as the representation of this genotype in provoked and unprovoked deep vein thrombosis (27% vs. 23%, p = 0.642), or in isolated deep vein thrombosis compared to pulmonary thromboembolism (25% vs. 33%, p = 0.735). Finaly, our results show that suppressed fibrinolytic functionality threefold increases risk of venous thrombosis (OR 3.02, CI 1.26-7.22), elevated levels of PAI-1 have no effect on the risk of deep vein thrombosis, as evidenced by OR of 0.86 with CI 0.59-1.25, elevated levels of t-PA antigen do not affect the risk of deep venous thrombosis (OR 1.53; CI 0.79-2.94), but increased concentration of TAFI increases more than twice this risk (OR 2.25; CI 1.16-4.35). PAI-1 4G/4G genotype does not affect venous thrombotic risk (OR 0.57; CI 0.27-1.20). Based on these results, we conclude that patients with deep vein thrombosis have suppressed fibrinolytic mechanism functionality compared to healthy subjects, the levels of t-PA antigen and plasminogen are significantly higher in patients with provoked venous thrombosis than in healthy subjects, there is no difference in PAI-1 concentration in patients with venous thrombosis and healthy persons, but the patients with deep vein thrombosis, regardless of its localisation or the type have a significantly higher level of TAFI as compared with healthy subjects. In addition, we can conclude that there is no difference in the prevalence of 4G/5G polymorphism in patients with venous thrombosis and healthy persons. Finally, we can say that suppressed fibrinolytic mechanism functionality threefold increases risk of deep vein thrombosis, elevated level of TAFI-a double increases this risk, while PAI-1 4G/5G polymorphism has no influence on the risk of venous thromboembolism.</p>

L’activateur tissulaire du plasminogène (tPA) est une sérine protéase qui est synthétisée principalementpar les cellules endothéliales des vaisseaux. Initialement découvert dans le compartiment vasculaire où sa fonctionprincipale est de participer à la fibrinolyse, le tPA est aussi exprimé dans le parenchyme cérébral par plusieurstypes cellulaires comme les neurones ou les oligodendrocytes. Le tPA est impliqué dans de nombreuses fonctionscérébrales comme la plasticité synaptique ou encore la potentialisation glutamatergique. Le tPA est aussi un acteurclé de la neuroinflammation. Il active la microglie et participe à l’ouverture de la barrière hémato-encéphalique pardes effets de type cytokine et via son domaine protéase en générant de la plasmine à partir du plasminogène. Uneactivité plus importante du tPA est retrouvée dans le liquide céphalorachidien des patients atteints de sclérose enplaques (SEP). De plus, le tPA possède des aspects délétères dans son modèle murin, l’encéphalomyélite autoimmune expérimentale (EAE). Dans le but de mieux comprendre le rôle du tPA dans la physiopathologie de l’EAE,nous nous sommes intéressés à son implication dans la réponse immunitaire pendant la maladie. Nos donnéesmontrent que les animaux tPA-/- ont des scores cliniques moins importants que les animaux WT pendant une EAE.Les nombres absolus de LT CD4+, de microglie activée et de macrophages infiltrés, ainsi que de cellulesdendritiques sont moins importants dans le parenchyme spinal des animaux tPA-/- en comparaison avec lesanimaux WT. En lien avec ces observations in vivo, le tPA augmente in vitro l’activation et la prolifération des LTainsi que la sécrétion d’IL-6 par un mécanisme dépendant du domaine protéase et de la génération de plasmine.Dans des expériences in vitro en collaboration avec l’équipe du Dr Diego Clemente, le tPA induit l’augmentation del’expression des molécules du CMH de classe II et des molécules de costimulation à la surface des cellulesdendritiques et des macrophages par un effet de type cytokine, suggérant une capacité plus importante pour cescellules à présenter des antigènes en présence de tPA. Notre étude apporte une meilleure compréhension du rôledu tPA dans la réponse immunitaire pendant l’EAE et ouvre de nouvelles perspectives dans l’étude de l’axe tPA/plasmine dans la physiopathologie de la maladie. / Tissue plasminogen activator (tPA) is a serine protease, mainly synthesized by endothelial cells of vessels.Initially discovered in the vascular compartment where its main function is to participate to fibrinolysis, tPA is alsopresent in the cerebral parenchyma, and expressed by several cell types like neurons or oligodendrocytes. tPA isinvolved in many physiological brain functions such as synaptic plasticity or glutamatergic potentiation. tPA is alsoa main actor of neuroinflammation. It activates microglia and participates in the opening of the blood-brain barrier(BBB) by cytokine-like effects and via its protease domain and plasmin generation from plasminogen. Interestingly,tPA activity is more important in cerebrospinal fluid of multiple sclerosis (MS) patients. In addition, tPA revealsdeleterious aspects in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. In order tobetter understand the role of tPA in EAE physiopathology, we focused on its involvement in the immune responseduring disease. tPA-/- EAE animals present less severe clinical scores than WT animals. Our results indicate alsothat absolute numbers of CD4 + T cells, activated microglia and infiltrated macrophages, as well as dendritic cellsare less important in the spinal parenchyma of tPA-/-. In connection with these in vivo observations, our in vitro datashow that tPA increases activation and proliferation of T cells, as well as IL-6 secretion by a protease-dependentmechanism and plasmin generation. In experiments in collaboration with Dr Diego Clemente's team, our data showthat tPA increases the expression of MHC class II and costimulatory molecules on the surface of dendritic cells andmacrophages in vitro by a cytokine-like effect, suggesting a more important ability for these cells to present antigenswith tPA. Our study provides a better understanding of the role of tPA in immune response during EAE, and opensup new perspectives in the study of the tPA / plasmin axis in the physiopathology of the disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Plasma membranes are complex supramolecular assemblies comprised of lipids and membrane proteins. Both types of membrane constituents are organized in highly dynamic patches with profound impact on membrane functionality, illustrating the functional importance of plasma membrane fluidity. Exemplary, dynamic processes of membrane protein oligomerization and distribution are of physiological and pathological importance. However, due to the complexity of the plasma membrane, the underlying regulatory mechanisms of membrane protein organization and distribution remain elusive. To address this shortcoming, in this thesis work, different mechanisms of dynamic membrane protein assembly and distribution are examined in a polymer-tethered lipid bilayer system using comple-mentary confocal optical detection techniques, including 2D confocal imaging and single molecule-sensitive confocal fluorescence intensity analysis methods [fluorescence correlation spectroscopy (FCS) autocorrelation analysis and photon counting histogram (PCH) method]. Specifically, this complementary methodology was applied to investigate mechanisms of membrane protein assembly and distribution, which are of significance in the areas of membrane biophysics and cellular mechanics. From the membrane biophysics perspective, the role of lipid heterogeneities in the distribution and function of membrane proteins in the plasma membrane has been a long-standing problem. One of the most well-known membrane heterogeneities are known as lipid rafts, which are domains enriched in sphingolipids and cholesterol (CHOL). A hallmark of lipid rafts is that they are important regulators of membrane protein distribution and function in the plasma membrane. Unfortunately, progress in deciphering the mechanisms of raft-mediated regulation of membrane protein distribution has been sluggish, largely due to the small size and transient nature of raft domains in cellular membranes. To overcome this challenge, the current thesis explored the distribution and oligomerization of membrane proteins in raft-mimicking lipid mixtures, which form stable coexisting CHOL-enriched and CHOL-deficient lipid domains of micron-size, which can easily be visualized using optical microscopy techniques. In particular, model membrane experiments were designed, which provided insight into the role of membrane CHOL level versus binding of native ligands on the oligomerization state and distribution of GPI-anchored urokinase plasminogen activator receptor (uPAR) and the transmembrane protein αvβ3 integrin. Experiments on uPAR showed that receptor oligomerization and raft sequestration are predominantly influenced by the binding of natural ligands, but are largely independent of CHOL level changes. In contrast, through a presumably different mechanism, the sequestration of αvβ3 integrin in raft-mimicking lipid mixtures is dependent on both ligand binding and CHOL content changes without altering protein oligomerization state. In addition, the significance of membrane-embedded ligands as regulators of integrin sequestration in raft-mimicking lipid mixtures was explored. One set of experiments showed that ganglioside GM3 induces dimerization of α5β1 integrins in a CHOL-free lipid bilayer, while addition of CHOL suppresses such a dimerization process. Furthermore, GM3 was found to recruit α5β1 integrin into CHOL-enriched domains, illustrating the potential sig-nificance of GM3 as a membrane-associated ligand of α5β1 integrin. Similarly, uPAR was observed to form complexes with αvβ3 integrin in a CHOL dependent manner, thereby causing the translocation of the complex into CHOL-enriched domains. Moreover, using a newly developed dual color FCS and PCH assay, the composition of uPAR and integrin within complexes was determined for the first time. From the perspective of cell mechanics, the characterization of the dynamic assembly of membrane proteins during formation of cell adhesions represents an important scientific problem. Cell adhesions play an important role as force transducers of cellular contractile forces. They may be formed between cell and extracellular matrix, through integrin-based focal adhesions, as well as between different cells, through cadherin-based adherens junctions (AJs). Importantly, both types of cell adhesions act as sensitive force sensors, which change their size and shape in response to external mechanical signals. Traditionally, the correlation between adhesion linker assembly and external mechanical cues was investigated by employing polymeric substrates of adjustable substrate stiffness containing covalently attached linkers. Such systems are well suited to mimic the mechanosensitive assembly of focal adhesions (FAs), but fail to replicate the rich dynamics of cell-cell linkages, such as treadmilling of adherens junctions, during cellular force sensing. To overcome this limitation, the 2D confocal imaging methodology was applied to investigate the dynamic assembly of N-cadherin-chimera on the surface of a polymer-tethered lipid multi-bilayer in the presence of plated cells. Here, the N-cadherin chimera-functionalized polymer-tethered lipid bilayer acts as a cell surface-mimicking cell substrate, which: (i) allows the adjustment of substrate stiffness by changing the degree of bilayer stacking and (ii) enables the free assembly of N-cadherin chimera linkers into clusters underneath migrating cells, thereby forming highly dynamic cell-substrate linkages with remarkable parallels to adherens junctions. By applying the confocal methodology, the dynamic assembly of dye-labeled N-cadherin chimera into clusters was monitored underneath adhered cells. Moreover, the long-range mobility of N-cadherin chimera clusters was analyzed by tracking the cluster positions over time using a MATLAB-based multiple-particle tracking method. Disruption of the cytoskeleton organization of plated cells confirmed the disassembly of N-cadherin chimera clusters, emphasizing the important role of the cytoskeleton of migrating cells during formation of cadherin-based cell-substrate linkages. Size and dynamics of N-cadherin chimera clusters were also analyzed as a function of substrate stiffness.

<p> Inherited bleeding disorders can be caused by mutations affecting platelet, coagulation, or fibrinolytic proteins. Quebec platelet disorder (QPD) is a rare, autosomal dominant disorder associated with increased expression of the fibrinolytic enzyme urokinase plasminogen activator (uPA) in platelets. Individuals with QPD experience delayed-onset bleeding after hemostatic challenges that is attenuated with fibrinolytic inhibitor therapy. The aims of this thesis were to: 1) determine if increased platelet uPA contributes to QPD clot lysis in vitro; 2) investigate whether QPD individuals have increased urinary uPA, as some individuals experience hematuria; and 3) map the genetic locus of QPD, and look for the putative mutation. Studies of clot lysis indicated that QPD platelets induce a gain-of-function defect in fibrinolysis when platelets are incorporated into clots. This suggests that accelerated fibrinolysis may contribute to QPD bleeding. Studies of urinary uPA in QPD showed that uPA is not increased, indicating that hematuria in QPD is likely a consequence of increased platelet uPA. This finding also suggests that uPA overexpression in QPD may be megakaryocyte-specific. Linkage studies showed that QPD is strongly linked to a 2 megabase region on chromosome 10 that harbors the uPA gene, PLA U. No mutations in PLA U or its regulatory regions were identified; however, a common haplotype for a 32.5 kilobase region around PLA U, including inheritance of a rare, linked polymorphism, suggests this is the most likely locus for QPD. mRNA studies in QPD platelets showed that QPD selectively increases expression of the linked PLAU allele, without similar increases in megakaryocyte progenitors or in saliva. These findings implicate a cis-mutation near PLA U as the cause of QPD. This thesis provides novel insights on the fibrinolytic abnormality in QPD blood, and on the QPD genetic locus. which will be important for identifying the precise mutation that converts normally prohemostatic platelets to profibrinolytic cells. </p> / Thesis / Doctor of Philosophy (PhD)

Exocrine pancreatic cancer is the fifth leading cause of death from malignant disease in Western society and it is one of the most aggressive human tumors. Once diagnosed, the 12-month patient survival is less than 5%. More than 90% of human exocrine tumors are classified as "ductal adenocarcinomas" on the basis of their microscopic appearance. The plasminogen system plays a critical role in intravascular thrombolysis as well as in other biological processes that require cellular migration, such as angiogenesis, inflammatory reactions, tissue remodelling, and tumor progression. There are two types of plasminogen activators that catalyze plasmin generation from plasminogen: tissue-type (tPA) and urokinase-type (uPA). Activation of plasminogen to plasmin results in progressive degradation of fibrin and other extracellular matrix components and may also lead to activation of metalloproteases, latent growth factors, and proteolysis of membrane glycoproteins. All these processes may contribute to tumor development and metastasis. There is extensive evidence supporting the notion that the uPA system, including its receptor and plasminogen activator inhibitor PAI-1, can contribute to tumorigenesis in a variety of tissue types but there is less evidence for such a role regarding tPA and annexin A2 (AnxA2), a putative tPA receptor. Previous studies of our group have shown that tPA is commonly expressed in pancreas cancer tissues and cell lines and appears to be selectively associated with the neoplastic phenotype. Using neutralizing antibodies or chemical inhibitors leads to reduced in vitro tumor invasion. Our results support that - in the pancreas - the tPA system plays an important role in tumor development and/or progression whereas the uPA system may play a more dominant role in pancreatitis. More recent studies have shown that tPA stimulates cell proliferation and angiogenesis in exocrine pancreatic tumors. These results allow new approaches to improve the treatment of this disease, but to do so it is necessary to use of mouse models of disease. In attempt to explore the role of tPA and its receptor, annexin A2, in pancreatic tumorigenesis, we have taken advantage of two well established transgenic mouse models: Ela1-TAg (1-127) and Ela1-myc. In these mice, transgenes are targeted to acinar cells using the Elastase-1 enhancer/promoter. We have also analyzed the pancreas of Ela1-CCK2 and MT-TGFa transgenic mice, as models of acinar-ductal transdifferentiation and ductal complex formation. Our results show that expression of tPA is undetectable in the non-neoplastic pancreatic epithelium and in metaplastic ducts and in acinar tumors. By contrast, tPA is overexpressed in neoplastic pancreatic ducts. This pattern expression is in agreement with the results described in humans, indicating that mouse models of pancreatic cancer may be useful for the study of human pathology. On the other hand, AnxA2 is undetectable in acinar tumors but it is detected in the apical membrane of normal and metaplastic duct epithelium. In addition, AnxA2 is strongly expressed in ductal tumor cells where it shows a non-polarized distribution. These results suggest that different molecular events may participate in the activation of tPA and its receptor, AnxA2, in non-neoplastic ducts. In order to analyze the role of tPA in the progression of pancreatic tumors, we mated Ela1-TAg and Ela1-myc transgenic mice to tPA-deficient mice. The proportion of tumors displaying pure acinar differentiation or mixed acinar/ductal components was similar in both mouse strains, indicating that tPA is not required for in acinar-ductal transdifferentiation. However, it was observed an increased survival in hybrid mice Ela1-myc:tPA-/- supporting a critical role for tPA in the progression of pancreatic ductal tumors. To get insight into the mechanism by wich tPA participates in this process we have analyzed factors related to tumor progression: tumors arising in a tPA-/- genetic background show a lesser vessel density and proliferation rate than those arising in wild type mice. These results indicate that tPA could play a role in angiogenesis stimulation and cell proliferation and suggest that the increase in survival observed in Ela1-myc tPA-/- mice could be a consequence of the inhibition of tumor angiogenesis and cell proliferation. In addition, we have analyzed the differential gene expression between Ela1-myc and Ela1-myc tPA-/- mice by microarrays. This analysis has led to the identification of related genes with tumor progression and invasion that can be a target for the action of tPA, although more work is necessary to determined their role. Finally, we have studied the direct effects of the expression of tPA in the pancreas, by the generation of two transgenic mice which tPA expression is targeted to acinar cells, using the Elastase-1 enhancer/promoter (Ela1-tPA), or to ductal cells using the Citokeratin 19 promoter (CK19-tPA). The results in Ela1-tPA mice show that overexpression of tPA in acinar cells does not affect normal mouse development. The effects on the pancreas analysed are currently being analyzed in greater detail. Altogether, the data described here support the relevant role of tPA in pancreas cancer progression and indicate that mouse models of pancreatic cancer may be useful for the preclinical evaluation of drugs targeting the tPA system.

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