Abstract : The chymase-dependant pathway responsible for converting Big ET-1 to ET-1 was established in vitro. It has only been recently, in 2009, that our group demonstrated that the conversion of Big ET-1 to ET-1 (1-31) can occur in vivo in mice (Simard et al., 2009), knowing that ET-1 (1-31) is converted to ET-1 via NEP in vivo (Fecteau et al., 2005). In addition, our laboratory demonstrated in 2013 that mMCP-4, the murine analogue of human chymase, produces ET-1 (1-31) from the Big ET-1 precursor (Houde et al. 2013). Thus far, in the literature, there are no specific characterizations of recombinant chymases (human or murine). In fact, the group of Murakami published in 1995 a study characterizing the CMA1 (human chymase) in a chymostatin-dependent fashion, using Angiotensin I as a substrate (Murakami et al., 1995). However, chymostatin is a non-specific inhibitor of chymase. It has been shown that chymostatin can inhibit elastase, an enzyme that can convert Angiotensin I to Angiotensin II (Becari et al., 2005). Based on these observations, the proposed hypothesis in the present study suggests that recombinant as well as extracted CMA1 from LUVA (human mast cell line), in addition to soluble fractions of human aortas, convert Big ET-1 into ET-1 (1-31 ) in a TY-51469 (a chymase-specific inhibitor) sensitive manner. In a second component, we studied the enzyme kinetics of CMA1 with regard to the Big ET-1 and Ang I substrate. The affinity of CMA1 against Big ET-1 was greater compared to Ang I (KM Big ET- 1: 12.55 μM and Ang I: 37.53 μM). However, CMA1 was more effective in cleaving Ang I compared to Big ET-1 (Kcat / KM Big ET-1: 6.57 x 10-5 μM-1.s-1 and Ang I: 1.8 x 10-4 ΜM-1.s- 1). In a third component involving in vivo experiments, the pressor effects of Big ET-1, ET-1 and Ang I were tested in conscious mMCP-4 KO mice compared to wild-type mice. The increase in mean arterial pressure after administration of Big ET-1 was greater in wild-type mice compared to mMCP- 4 KO mice. This effect was not observed after administration of ET-1 and / or Ang I. / Résumé : La voie de conversion de Big ET-1 en ET-1, chymase dépendante a été établie in vitro. Ce n'est que récemment, en 2009 que notre groupe a démontré que la conversion de Big ET-1 en ET-1 (1-31) peut avoir lieu in vivo chez la souris (Simard et al., 2009), sachant que ET-1 (1-31) est convertie en ET-1 via NEP in vivo (Fecteau et al., 2005). En plus, en 2013, notre laboratoire a démontré que la mMCP- 4, l'analogue murin de la chymase humaine, produit l'ET-1 (1-31) à partir du précurseur Big ET-1 (Houde et al., 2013). Jusqu'a présent, dans la littérature, on ne trouve pas de caractérisations spécifiques de chymases (humaine ou murine) recombinantes. En fait, le groupe de Murakami, en 1995, a publié une étude caractérisant, d'une façon chymostatin dépendante, la CMA1 (chymase humaine) en utilisant l'Angiotensine I comme substrat (Murakami et al., 1995). Cependant, le chymostatin est un inhibiteur non-spécifique de la chymase. Il a été démontré que le chymostatin peut inhiber l'élastase, une enzyme pouvant convertir l'Angiotensine I en Angiotensine II (Becari et al., 2005). Basé sur ces observations, l'hypothèse formulée dans la présente étude est que la CMA1 recombinante ou extraite des cellules LUVA (lignée humaine de mastocytes) ou des fractions solubles des aortes humaines convertit la Big ET-1 en ET-1 (1-31) d'une façon TY-51469 (un inhibiteur spécifique de la chymase) sensible. Dans un deuxième volet, on a étudié la cinétique enzymatique de CMA1 en vers le substrat Big ET-1 et Ang I. L’affinité de CMA1 contre la Big ET-1 était plus grande comparé à l’Ang I (KM Big ET-1 : 12.55 μM et Ang I : 37.53 μM). Cependant CMA1 était plus efficace dans le clivage de l’Ang I comparé à la Big ET-1 (Kcat/KM Big ET-1 : 6.57 x 10-5 μM-1 .s-1 et Ang I : 1.8 x 10-4 μM-1 .s-1 ). Dans un troisième volet impliquant des expériences in vivo, l’effet presseur de la Big ET-1, l’ET-1 et l’Ang I a été testé chez des souris conscientes mMCP- 4 KO comparé à des souris de type sauvage. L’augmentation de la pression artérielle moyenne a été plus importante chez les souris de type sauvage après l’administration de Big ET-1 que chez les souris mMCP-4 KO. Cet effet n’a pas été observé après l’administration d’ET-1 et/ou d’Ang I ce qui explique le rôle de la chymase dans l’effet de la conversion de Big ET-1 en ET-1 (1-31).

Mast cells play a critical role in both acute and chronic inflammation and mature in peripheral tissues from bone marrow-derived progenitors that circulate in the blood as immature precursors. Mast cell progenitors are likely to encounter the serum-borne bioactive sphingolipid metabolite, sphingosine-1-phosphate (S1P), during migration to target tissues. Mast cells developed from human cord blood-derived progenitors cultured with stem cell factor (SCF) alone express intragranular tryptase (MCT), the phenotype predominant in the lung. S1P accelerated the development of cord blood-derived mast cells (CB-MCs) and strikingly increased the numbers of mast cells expressing chymase. These mast cells have functional FcepsilonRI, and similar to skin mast cells that express both tryptase and chymase (MCTC), also express CD88, the receptor for C5a, and are activated by anaphylatoxin C5a and the secretagogue compound 48/80. S1P induced release of IL-6, a cytokine known to promote development of functionally mature MCTC, from cord blood cultures containing adherent macrophages, and from highly purified macrophages, but not from macrophage-depleted CB-MCs. In contrast, S1P stimulated secretion of the chemokine, monocyte chemoattractant protein 1 (MCP-1/CCL2), from these macrophage-depleted and purified CB-MCs.

Mast cells are key effector cells in allergic and inflammatory diseases. However, their primary role is most likely in host defence against parasitic and bacterial infections. Mast cells are a particularly rich source of serine proteases. These proteases belong to the chymase or the tryptase family, which are encoded from the mast cell chymase and the multigene tryptase loci, respectively. To better understand the biological functions and the molecular evolution of these enzymes we have studied the organisation of these two loci in species ranging from fish to human. We show that the mast cell chymase locus has evolved from a single founder gene to a complex locus during the past 200 Myr of mammalian evolution. Forty-five fish candidate genes for hematopoietic serine proteases were also identified. However, in phylogenetic analyses none of them grouped with individual branches holding mammalian mast cell chymase locus genes, indicating an independent parallel evolution in fish. Studies of the evolution of the multigene tryptase locus showed that this locus has been highly conserved between marsupials and eutherians. However, no genes belonging to the individual subfamilies identified in eutherians could be identified in fish, amphibians or in birds, which also here indicates parallel evolution. To study the evolution of specific cleavage specificities associated with these proteases, the extended cleavage specificity of opossum α-chymase was determined and found to be nearly identical to human mast cell chymase and the major mouse mast cell chymase mMCP-4. This indicates a strong pressure to maintain this specificity during mammalian evolution. Basophils are rare blood cells with functions similar to mast cells that when mature almost completely lack mRNA. To study the proteome and to primarily characterize the granule protein content of basophils, an in vitro purification protocol was developed to obtain transcriptionally active umbilical cord blood-derived basophil precursors.

Submitted by HELENA RIBEIRO SOUZA null (helena.riber@hotmail.com) on 2016-08-11T15:45:49Z No. of bitstreams: 1 Dissertação Helena R Souza.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-12T19:10:30Z (GMT) No. of bitstreams: 1 souza_hr_me_sjrp.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) / Made available in DSpace on 2016-08-12T19:10:30Z (GMT). No. of bitstreams: 1 souza_hr_me_sjrp.pdf: 28021261 bytes, checksum: cfa8bae6ea9a632c8ff6462cc740385a (MD5) Previous issue date: 2016-07-29 / O processo de reparo de lesões térmicas pode ser dividido nas fases de inflamação, proliferação e remodelação. No seu processo de desgranulação, os mastócitos (MCs) liberam fatores quimiotáticos, citocinas e proteases, como triptase e quimase, para o meio extracelular, contribuindo na degradação da matriz lesada e síntese de uma nova. Além disso, estudos indicam que os MCs armazenam a proteína anti-inflamatória anexina A1 (AnxA1), envolvida na inflamação, apoptose, crescimento e diferenciação celular. Contudo, não se conhecem relatos da expressão e função da AnxA1 no reparo de queimaduras. Diante disso, os objetivos desse estudo foram quantificar e avaliar a ativação, acúmulo de histamina e heterogeneidade para triptase e quimase dos MCs, bem como, verificar a expressão da AnxA1, quantificar macrófagos, e dosar as citocinas TNF-α, IL-1β, IL-6, IL-10 e a quimiocina MCP-1 em modelo de queimadura de segundo grau em ratos Wistar. Para o desenvolvimento da lesão os animais foram anestesiados e submetidos à aplicação de um bloco metálico aquecido no dorso. Os animais foram divididos em grupos controles e tratados com pomada de sulfadiazina de prata a 1% (SDP 1%). As análises foram realizadas em 3, 7, 14 e 21 dias após injúria, e também em fragmentos de pele normal. Avaliações macroscópicas e histopatológicas da cicatrização confirmaram as características de queimadura de segundo grau e a melhor evolução da cicatrização nos animais tratados. A quantificação dos MCs mostrou grande quantidade de células intactas na pele normal e redução significante dessas células nas fases de inflamação (dia 3) e de proliferação (dia 7). Nas fases de proliferação de matriz (dia 14) e remodelação (dia 21) foi verificada maior quantidade de MCs nos animais controles e essas células foram observadas, principalmente, desgranuladas no dia 14 e intactas no dia 21. Ainda, no grupo tratado aos 7 dias e em ambos os grupos aos 14 dias, foi encontrada diferença entre MCs com grande quantidade de histamina e MCs totais. A análise da heterogeneidade dos MCs revelou maior número de MCs positvos para triptase (MCT) do que para quimase (MCQ) na pele normal e redução de MCTs e MCQs nas primeiras fases da cicatrização. Contudo, na fase de remodelação, muitos MCQs foram observados no grupo controle. A expressão da AnxA1 foi fraca na pele normal com aumento nas fases de inflamação e de proliferação. Aos 21 dias após lesão, a expressão da AnxA1 foi maior nos animais tratados com SDP 1%, no estroma e também em regiões epiteliais próximas à neogênese dos anexos cutâneos. As análises das citocinas mostraram aumento das pró-inflamatórias TNF-α, IL-1β e IL-6, e da citocina anti-inflamatória IL-10, nas fases iniciais do reparo, especialmente nos dias 3 e 7. O mesmo foi observado com relação a quimiocina MCP-1 e a quantificação de macrófagos. Nossos resultados evidenciaram diferenças no número de MCs e na imunomarcação da AnxA1 entre os grupos estudados, moduladas pelo tratamento com a SDP 1% e indicam que essas células e proteínas podem ser alvos terapêuticos no processo de regeneração de queimaduras. / The repair process of thermal injury can be divided into phases of inflammation, proliferation and remodeling. The mast cells (MCs) in their degranulation process, release chemotactic factors, cytokines and proteases, as tryptase and chymase, to the extracellular environment contributing to the degradation of damaged matrix and synthesis of a new one. Moreover, studies indicate that MCs store the antiinflammatory protein annexin A1 (AnxA1) which involved in inflammation, apoptosis, growth and cell differentiation. However, there are no known reports of the expression and function of AnxA1 in burns repair. Thus, the objectives of this study were to quantify, assess the activation, histamine accumulation and heterogeneity for tryptase and chymase of MCs, as well as, check the expression of AnxA1, associated with macrophages quantification and the levels of TNF-α, IL-1β, IL-6, IL-10 and MCP-1 in a second degree burn model in rats. For the development of the lesions, animals were anesthetized for applying a water-heated metal block in the dorso. The animals were divided into control and treated or not with the cream of silver sulfadiazine 1% (SDP 1%). The analyzes were performed on 3, 7, 14 and 21 days after injury, and also in normal skin fragments. Macroscopic and histopathological evaluations of healing confirmed the burning characteristics of second degree and the better progress of wound repair in treated animals. Quantification of MCs showed large amount of intact cells in normal skin and a significant reduction of these cells in the stages of inflammation (day 3) and proliferation (day 7). In the phases of matrix proliferation (day 14) and remodeling (day 21) it was observed increased amount of MCs, only in the control animals, and these cells were mainly degranulated on day 14, but intact on day 21. Also, in the treated group on day 7 and in both groups on day 14, it was found difference between MCs with large amounts of histamine and total MCs. The heterogeneity analysis revealed more MCs positves for tryptase (MCT) than for chymase (MCC) on normal skin and reduced MCTs and MCQs in the early stages of healing. However, in the remodeling phase, many MCCs were observed in the control group. The expression of AnxA1 was low in normal skin and increased in the stages of inflammation and proliferation. On 21 days after injury, the expression of AnxA1 was higher in animals treated with SDP 1% in the stroma and epithelial cells especially in regions close to neogenesis of skin attachments. Analysis of the inflammatory mediators showed an increase pro-inflammatory TNF-α, IL-1β and IL-6 and anti-inflammatory IL-10 cytokines in the early stages of the repair, especially on days 3 and 7. The same was observed for MCP-1 chemokine and quantification of macrophages. Our results showed differences in the number of MCs and AnxA1 expression between groups, that were modulated by treatment with the SDP 1%, indicating that these cells and protein can be used as therapeutic targets in burns regeneration process.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-12-19T17:52:15Z No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 740997 bytes, checksum: fec1f12612ff6a41fbbbee9640600a35 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Júlio, falta o arquivo da tese. on 2016-12-26T12:31:54Z (GMT) / Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2017-01-02T15:41:57Z No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-01-03T09:40:55Z (GMT) No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-01-03T09:40:55Z (GMT). No. of bitstreams: 2 Tese - Maurício Barcelos Costa - 2014.pdf: 10731375 bytes, checksum: d59433128feb9bc7facacb81cb311a98 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-02-26 / Leprosy is a complex, chronic, infectious dermato-neurological disease that affects the skin and peripheral nerves especially during acute immune-inflammatory episodes known as type 1/T1R and type 2/T2R reactions. There is no experimental model for leprosy and leprosy skin lesions have been extensively used to unravel its multifaceted immunopathological mechanisms.This study investigated in situ expression of two distinct cell populations with important immunoregulatory roles: T regulatory (Treg) cells and mast cells (MC) in diverse skin diseases with emphasis on leprosy T1R and T2R. For the Treg cell study, 154 skin biopsies from 114 participants belonging to 3 groups were investigated: 1. Leprosy (n=74), 56 T1R (28-paired biopsies reactionfree/reactional from the same patient, 28 single reactional biopsy), 18 T2R (12 pairedreaction-free/reactional lesions, 6 single reactional biopsy); 2. Dermatoses: (n=29) noninfectious and cutaneous infectious diseases; 3. Normal controls: skin fragment of mammoplasty from healthy females that had cosmetic surgery. Double immunohistochemical detection of Treg cells was performed with automated platform for CD25 and Foxp3 staining. Quantifications of double immunostained Treg cells was performed (values expressed by mm2 ) blinded to the participants’ clinical status. For the mast cell study 80 skin biopsies from 3 groups were investigated: 40 newly diagnosed untreated leprosy patients (18 reaction-free, 11 T1R, 11 T2R), 29 patients with other dermatoses (the same as for Treg study) and 11 normal skins. Toluidine blue stained intact and degranulated MC counts/mm2 ; streptavidin-biotin-peroxidase immunostaining was used to detect tryptase/try+ and chymase/chy+ MCs and their density (median optical density) was evaluated. Results: Treg study: Not one CD25+ Foxp3+ Treg cell was seen in any of the 11 normal skin sections while variable numbers were detected in skin diseases (p<0.0001); the number of double stained cells was higher in infectious compared to non-infectious diseases (p=0.008). Treg cell numbers were comparable between leprosy and other infectious dermatoses (p=0.157) Treg cell counts in reactional lesions were higher than in reaction-free leprosy lesions (p<0.002). Paired biopsies of T1R or T2R reactional/reaction-free lesions showed xxvii increased numbers of Treg during T1R compared to reaction-free lesions from the same patient (p< 0.001). Treg cell median in T1R developed during MDT was slightly higher compared to T1R developed at diagnosis in naïve patients (p=0.047). There was a trend in increasing Treg cell numbers from the tuberculoid to borderline-lepromatous form, which showed the highest median value of Tregs, however this difference was not significant (p>0.8). Mast cell study: Infectious and non-infectious skin lesions showed higher numbers of degranulated than intact MC both for leprosy and other dermatoses, compared to normal skin. The numbers of degranulated MC were higher than intact MC regardless of the leprosy form (from tuberculoid/TT to lepromatous/LL), regardless of the occurrence of leprosy reactions (reactional and reaction-free) and regardless of the type of reaction (T1R/T2R). Try+ MC numbers and density were higher than chy+ MC in leprosy, in reaction-free and reactional lesions, particularly in T2R, but not in other dermatoses. Conclusions: Higher Treg numbers seen in T1R suggest Treg role in suppressing the exacerbated cell-mediated phenomenon that causes T1R. Differential expression/ of try+ and chy+ MC subsets was seen in leprosy compared to other skin diseases and to normal skin. However, neither leprosy form nor leprosy reaction was associated with MC changes in lesions suggesting that the Mycobacterium leprae infectious process per se dictates MC expression in leprosy skin lesions. / A hanseníase é uma complexa doença dermato-neurológica, crônica, de causa infecciosa que afeta a pele e os nervos periféricos, especialmente durante os episódios imunoinflamatórios agudos conhecidos com reações tipo 1/RT1 e tipo 2/RT2. Não existe modelo experimental para hanseníase e as lesões de pele têm sido extensivamente usadas para desvendar os multifacetados mecanismos imunopatológicos associados com a doença. Este estudo investigou a expressão in situ de duas distintas populações celulares que apresentam importante papel imunorregulatório: células Treg (Treg) e mastócitos (MC) em pele normal e diversas doenças cutâneas com ênfase nas reações hansênicas RT1 e RT2. Para o estudo de Tregs foram utilizadas 154 biópsias cutâneas de 114 participantes de três grupos: 1. Hanseníase (n=74), 56 RT1 (28-biópsias pareadas do mesmo paciente sem reação/durante reação, 28 biópsias únicas de RT1), 18 RT2 (12 biópsias pareadas sem reação/durante reação, 6 biópsias únicas de RT2); 2. Dermatoses: (n=29) doenças cutâneas não infecciosas e infecciosas. Controles Normais: fragmentos de peles obtidos de mamoplastias eletivas em mulheres saudáveis. Imunomarcação dupla CD25+ Foxp3+ de células Treg foi realizada em plataforma automatizada. Quantificação das células Treg duplo positivas foram feitas sem conhecimento da condição clínica do paciente (valores expressos em mm2 ). Para o estudo dos MC 80 biópsias de 3 grupos foram investigadas: 40 pacientes com hanseníase recém diagnosticados não tratados (18 sem reação, 11 RT1, 11 RT2), 29 pacientes com outras dermatoses e 11 biópsias de pele normal. Quantificação de MC intactos e desgranulados corados por azul de toluidina/mm2 ; imunomarcação streptavidina-biotina-peroxidase foi empregada para detectar MC triptase/try+ e chimase/chy+ e a densidade ótica (mediana da densidade ótica) foi avaliada. Resultados: Estudo das Tregs: Nenhuma célula Treg CD25+ Foxp3+ foi identificada em nenhuma das 11 amostras de pele normal, enquanto um número variável de Tregs foi identificado nas diversas doenças cutâneas (p<0.0001); o número de células Treg duplo positivas foi maior nas doenças infecciosas comparado com as não-infecciosas (p=0.008). Medianas de Treg entre hanseníase e outras doenças infecciosas foram semelhantes (p=0.157). Quantificação de Tregs em lesões reacionais foram maiores do que as sem reação (p<0.02). Nas biópsias pareadas de lesões de xxv RT1/sem reação ou RT2/sem reação, números maiores de Treg foram vistos durante a RT1 quando comparados com a não reacional do mesmo paciente (p< 0.001). Mediana de Treg em RT1 desenvolvidas durante MDT foi ligeiramente superior comparada a RT1 desenvolvida em pacientes sem de tratamento (p=0.047). Observou-se uma tendência de aumento no número das Tregs do polo tuberculoide em direção ao lepromatoso, mais especificamente até a forma borderline-lepromatosa que apresentou a maior mediana da quantificação de Treg, entretanto esta diferença não foi estatisticamente significativa (p>0.8). Estudo dos Mastócitos: lesões cutâneas de origem infecciosa e não infecciosa mostraram números aumentados de mastócitos desgranulados do que intactos tanto na hanseníase como nas outras dermatoses quando comparados com pele normal. Os números de mastócitos (MC) desgranulados foram maiores do que intactos, independente da forma de hanseníase (do polo tuberculoide/TT ao lepromatoso/LL), independente da ocorrência de reações hansênicas (lesão reacional/sem reação) e independente do tipo de reação (RT1/RT2). Número e densidade de mastócito triptase positivo (MC try+ ) estão aumentados em relação aos quimase positivo (MC chy+ ) na hanseníase, em pacientes com e sem reação, particularmente na RT2, mas não nas outras dermatoses. Conclusões: aumento nas Treg detectados durante RT1 sugerem papel supressor dessas células em eventos associados à resposta imune celular exacerbada, responsáveis pela RT1. Expressão diferencial das subpopulações de MC try+ e chy+ foi observada na hanseníase em relação a outras doenças cutâneas e pele normal. Entretanto, nem a forma da hanseníase, nem a ocorrência de reação hansênica, estava associada a mudanças nas subpopulações de MC nas lesões sugerindo que o processo infeccioso pelo Mycobacterium leprae per se direciona a expressão de MC nas lesões cutâneas da hanseníase.

Spiral artery (SA) remodeling is a fundamental process during early pregnancy that involves the action of vascular smooth muscle cells (VSMCs), maternal immune cells, but also fetal extravillous trophoblast cells (EVTs) and the extracellular matrix (ECM). Mast cells (MCs) and their mediator chymase, have been identified as prominent players for a sufficient SA remodeling process at the fetal-maternal interface in vivo. In contrast, endocrine disrupting chemicals (EDC), especially Bisphenol A (BPA) and 17-α-ethinylestradiol (EE2), have been shown to have a negative impact on SA remodeling in animal models in vivo. However, neither the mechanisms underlying the positive effects of MCs and chymases for the remodeling process, nor the interference of EDCs on SA remodeling, have been elucidated. The purpose of the present work was to evaluate the effect of MCs and recombinant human chymase CMA1 (rhuCMA1) on the phenotype and/or behavior of VSMCs, EVTs and ECM in vitro. Moreover, the influence of BPA and EE2 on the functionality of EVTs in vitro was observed. Using an immortalized human trophoblast cell line (HTR8/SV-neo), a mouse trophoblast cell line (SM9-2), human primary uterine vascular smooth muscle cells (HUtSMCs) and mouse primary uterine smooth muscle cells (SMCs), we assessed the effects of the human MC cell line HMC-1, the mouse mast cell line MC/9 and rhuCMA1 on VSMCs, EVTs and ECM. Additionally, the HTR- 8/SV-neo cells functionality was evaluated after treatment with BPA or EE2. We found that mouse MC/9 cells induce fibronectin expression and migration in SMCs. Furthermore MC/9 cells increase the proliferation and migration of SM9-2 cells. Both human HMC-1 cells and rhuCMA1 stimulate the migration, proliferation, and change of synthetic/contractile marker expression in HUtSMCs. In addition, HMC-1 cells increase the proliferation and migration of HTR8/SVneo trophoblast cells while having an impact on the expression of tissue remodeling genes. HTR8/SVneo cells presented increased migration rates along with decreased expression of the matrix-metalloproteinase regulator genes (TIMPs) upon treatment with rhuCMA1. Moreover, BPA interfered with HTR-8/SV-neo cell proliferation and reduced MMP2 expression in HTR-8/SV-neo. Interestingly, EE2 had no impact on proliferation or migration but suppressed the MMP2 expression in HTR-8/SV-neo cells. The obtained results indicate that MCs, and partly their mediator chymase CMA1, shape the phenotype and modulate the functionality of VSMCs and EVTs. Collectively, possible mechanisms by which MCs and specifically rhuCMA1 promote SA remodeling were identified. The findings are relevant for the understanding of this crucial step in pregnancy and thus, for the comprehension of dysregulated pathways that can lead to pregnancy complications like fetal growth restriction and preeclampsia. Moreover, this work contributes to the knowledge about how EDCs impact on early pregnancy and highlights the high risk of EDCs exposure disturbing the fundamental reproductive process of SA remodeling.

Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.

Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.

Spiral-artery (SA) remodeling is a fundamental process during pregnancy that involves the action of cells of the initial vessel, such as vascular smooth-muscle cells (VSMCs) and endothelial cells, but also maternal immune cells and fetal extravillous trophoblast cells (EVTs). Mast cells (MCs), and specifically chymase-expressing cells, have been identified as key to a sufficient SA remodeling process in vivo. However, the mechanisms are still unclear. The purpose of this study is to evaluate the effects of the MC line HMC-1 and recombinant human chymase (rhuCMA1) on human primary uterine vascular smooth-muscle cells (HUtSMCs), a human trophoblast cell line (HTR8/SV-neo), and human umbilical-vein endothelial cells (HUVEC) in vitro. Both HMC-1 and rhuCMA1 stimulated migration, proliferation, and changed protein expression in HUtSMCs. HMC-1 increased proliferation, migration, and changed gene expression of HTR8/SVneo cells, while rhuCMA treatment led to increased migration and decreased expression of tissue inhibitors of matrix metalloproteinases. Additionally, rhuCMA1 enhanced endothelial-cell-tube formation. Collectively, we identified possible mechanisms by which MCs/rhuCMA1 promote SA remodeling. Our findings are relevant to the understanding of this crucial step in pregnancy and thus of the dysregulated pathways that can lead to pregnancy complications such as fetal growth restriction and preeclampsia.