Neste estudo, avaliou-se o efeito da exposição intermitente à Ang II, levando em consideração uma dose que tenha uma ação não pressora, sobre a liberação de citocinas inflamatórias como a interleucina-6 (IL-6) e fator de crescimento transformante beta (TGFβ), bem como PAI-1 e plasminogênio/plasmina. O estudo foi realizado em camundongos machos C57Bl/6, submetidos ao tratamento com Ang II (30ng/kg), com losartan (30mg/kg) ou uma combinação destes, nos tempos de: 30 minutos, 1, 3 e 10 dias. As avaliações mostraram que a Ang II não altera pressão arterial, sugerindo que os aumentos observados de IL-6 e TGFβ sejam decorrentes de ação direta da Ang II. A Ang II promove aumento tanto agudo de TGFβ, possivelmente associado à ação proteolítica da plasmina e alterações vasculares transitórias compatíveis com aumento de permeabilidade, como crônico de TGFβ, possivelmente associado ao aumento da expressão gênica, levando ao aumento da deposição de colágeno vascular. / This study, evaluated the effect of the intermittent exposure to Angiotensin II (Ang II), taking in account a non-pressor dose on the release of inflammatory cytokines such as interleukin-6 (IL-6), transforming growth factor beta (TGFβ) as well PAI-1 and plasminogen /plasmin. The study was conducted on male mice C57BL/6 subjected to the treatment with angiotensin II (30 ng/kg), losartan (30 mg/kg) or a combination thereof, at times: 30 minutes, 1, 3 and 10 days. The evaluations showed that Ang II did not change blood pressure, suggesting that the increases of IL-6 and TGFβ may be by due to direct action of Ang II. Ang II promotes both acute increase of TGFβ, possibly associated with the proteolytic action of plasmin and transient vascular changes consistent with increased permeability, such as chronic increase of TGFβ, possibly associated with increased gene expression, leading to increased vascular collagen deposition.

Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams. / Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text]

Systemic sclerosis (SSc) – is one of the most complicated and fatal systemic diseases, and the lack of effective therapy is very evident. The tyrosine kinase inhibitor imatinib mesylate (IM) was shown to inhibit TGF-β and PDGF signaling pathways and prevent the development of dermal fibrosis upon challenge with bleomycin in murine model of SSc. The aim of therapy is not only to stop disease progression, but even induce regression of preexisting fibrosis. On other hand, blocking TGF-β and PDGF signaling in angiogenesis might worsen the vascular manifestations of SSc. We found important to evaluate effectiveness of IM for the treatment of pre-established tissue fibrosis and to exclude that the anti-fibrotic effects of IM are complicated by inhibitory effects on endothelial cell functions. Aim of the study: assess the effect of IM on the process of fibrosis and endothelium in experimental models of systemic sclerosis and cell cultures. Objectives of the study: assess the effectiveness of IM on murine models of established fibrosis; evaluate if IM has an effect on basal functions of endothelial cells; assess effect of IM on the process of angiogenesis. We have shown that IM exerts potent antifibrotic effects in two different models of SSc. Imatinib was effective for prevention of fibrosis and for treatment of established dermal fibrosis. We’ve demonstrated that IM does not inhibit major functions of endothelial cells. Thus, IM might not augment further the preexisting vascular... [to full text] / Sisteminė sklerozė (SSc) – viena sunkiausių ir fatališkiausių autoimuninių sisteminių reumatinių ligų, o bazinių vaistų stygius, šiai ligai gydyti, itin didelis. Į onkologinę klinikinę praktiką įdiegtas tirozinkinazių inhibitorius – imatinibo mezilatas(IM). IM blokuoja TGF-β ir PDGF intraląstelinio signalo perdavimą ir taip sąlygoja fibrozės prevenciją SSc pelių modelyje. Mums buvo svarbu išsiaiškinti, ar imatinibas gali turėti įtakos ne tik prevencijai, bet ir susiformavusiai fibrozei. Be to TGF-β ir PDGF blokavimas angiogenezėje, galėtų riboti daug žadančio fibrozės inhibitoriaus IM naudojimą gydant SSc. Darbo tikslas: įvertinti imatinibo mezilato poveikį fibrozės procesui ir endoteliui sisteminės sklerozės eksperimentiniuose modeliuose ir ląstelių kultūrose. Darbo uždaviniai: įvertinti imatinibo efektyvumą neuždegiminiame SSc modelyje ir patikrinti imatinibo mezilato efektyvumą uždegiminiame suformuotos fibrozės modelyje; ištirti, ar terapinės imatinibo mezilato koncentracijos daro neigiamą poveikį gyvybinėms endotelio funkcijoms; įvertinti imatinibo mezilato poveikį angiogenezės etapams. Mūsų gauti duomenys rodo, kad: IM ne tik sustabdė bet ir paskatino jau egzistuojančios (bleomicino sukeltos) odos firbrozės regresiją; IM ryškiai sumažino poodžio ir odos storį, bei normalizavo miofibroblastų skaičių Tsk-1 pelėse; IM neturėjo poveikio endotelio ląstelių bazinėms funkcijoms; IM neturėjo neigiamo poveikio angiogenezės etapams.

In der vorliegenden Arbeit konnten erstmals der TGF-β-Signalweg und dessen Smad- Signalmoleküle innerhalb der MPCs nachgewiesen werden. Dieser Nachweis erfolgte sowohl auf zellulärer und Gewebeebene als auch auf Gen- und Proteinebene. Zusätzlich konnte auf Gen- und Proteinebene gezeigt werden, dass die Signalmoleküle Smad2, Smad3 und Smad4 in MPCs aus gering erkranktem Meniskusgewebe eine vermehrte Expression aufweisen im Vergleich zu den MPCs aus hochgradig erkranktem Meniskusgewebe. Diese Erkenntnis weist auf eine mögliche protektive Funktion des TGF- β-Signalwegs während degenerativer Prozesse im Meniskusgewebe hin. Um die Effekte des TGF-β-Signalwegs und dessen Smad-Signalmoleküle genauer zu verstehen und besser beschreiben zu können, wurde eine Überexpression der Smad-Signalmoleküle innerhalb von MPCs durchgeführt und die Auswirkungen dieser auf die Kollagen I- und Kollagen II-Synthese genauer beleuchtet. Infolgedessen konnte sowohl eine vermehrte Kollagen I-Synthese als auch eine vermehrte Kollagen II-Synthese festgestellt werden. Dies bestätigt die Annahme, dass dem TGF-β-Signalweg und dessen Smad-Signalmolekülen eine zentrale, protektive Funktion während der Meniskusdegeneration zukommt. Durch die vermehrte Synthese von Matrixkomponenten wird den Degenerationsprozessen innerhalb des Meniskusgewebes entgegengewirkt. Ein nicht degenerierter bzw. ein regenerierter Meniskus besitzt eine biomechanische Schutzfunktion für das Kniegelenk und wirkt somit einer Kniegelenkarthrose entgegen. In Zukunft könnte der TGF-β-Signalweg  einen möglichen Ansatzpunkt für therapeutische Behandlungen bei Meniskusläsionen darstellen. Da Meniskusdefekte häufig direkt mit einer Osteoarthrose im Kniegelenk assoziiert sind, spielt die durch den TGF-β-Signalweg induzierte Regeneration von Meniskusgewebe auch in der Prävention der Osteoarthrose eine zentrale Rolle.

La Fibrose Pulmonaire idiopathique (FPI) est une maladie fibroproliférative pour laquelle il n’existe aucun traitement efficace. Les mécanismes à l’origine de cette pathologie sont méconnus et impliquent plusieurs types cellulaires et facteurs de croissance, comme le TGF-β responsable de la différenciation de fibroblastes en myofibroblastes. Pour mieux comprendre ces mécanismes physiopathologiques, nous nous sommes intéressés à l’implication des miARN dans ce processus. Une analyse par puces à ADN de l’ensemble des miARN modulés dans des échantillons pulmonaires de souris, résistantes ou sensibles à la fibrose pulmonaire induite par la bléomycine, nous a permis d’identifier miR-199a-5p comme le meilleur candidat associé à la fibrose pulmonaire mais aussi fibrose rénale et hépatique. J’ai ensuite démontré que l’expression de miR-199a-5p était induite par le TGF-β in vitro, et que sa surexpression ectopique induisait la différenciation des fibroblastes. Une combinaison d’approche in silico et expérimentale, m’a permis d’identifier la Cavéoline-1 (CAV-1) comme cible de ce miARN. La CAV-1 est impliquée dans la dégradation du récepteur TGF-β. Ainsi, l’inhibition de CAV-1 par miR-199a-5p constitue une boucle de rétrocontrôle positif exacerbant la voie TGF-β. De manière intéressante, l’inhibition de miR-199a-5p in vitro régule la différenciation, la prolifération et la migration des fibroblastes pulmonaires par le TGF-β. Par ailleurs, nos résultats précliniques indiquent que l’inhibition de ce miARN diminue les marqueurs de fibrose, permettant d’envisager le développement de nouvelles approches thérapeutiques dans le traitement de la FPI et d’autres maladies fibroprolifératives. / Idiopathic Pulmonary Fibrosis (IPF) is a fibroproliferative disease with poor prognosis and for which no effective treatment exists. The mechanisms of this disease remain poorly understood and involve numerous cell types and growth factors such as TGF-β, which leads to the activation of lung fibroblasts into myofibroblasts; the key cell type driving the fibrogenic process. In this context, we focused the involvement of miRNAs in fibrosis process. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to lung fibrosis after bleomycin exposure. We identified miR- 199a-5p as the best candidate associated with lung fibrosis but also kidney and liver fibrosis. I observed that miR-199a-5p expression was induced upon TGF-β exposure, and that its ectopic expression was sufficient to promote the pathogenic activation of pulmonary fibroblasts. Using combination of targets miRNA prediction tools and a transcriptomic approach we identified the Caveolin-1 (CAV-1), a critical mediator of pulmonary fibrosis, as a specific target of miR-199a-5p. Thus, we shown that miR-199a-5p is a key effector of TGF-β signaling in lung fibroblasts by regulating CAV1. Interestingly, inhibition of miR-199a-5p in vitro prevents the differentiation, proliferation and migration of fibroblasts after TGF-β stimulation. Finally, our preclinical results indicate that inhibition of this miRNA decreases fibrosis markers. Thus, miR-199a-5p behaves as a major regulator of tissue fibrosis with therapeutic potency for the treatment of IPF and fibroproliferative diseases.

Neste estudo, avaliou-se o efeito da exposição intermitente à Ang II, levando em consideração uma dose que tenha uma ação não pressora, sobre a liberação de citocinas inflamatórias como a interleucina-6 (IL-6) e fator de crescimento transformante beta (TGFβ), bem como PAI-1 e plasminogênio/plasmina. O estudo foi realizado em camundongos machos C57Bl/6, submetidos ao tratamento com Ang II (30ng/kg), com losartan (30mg/kg) ou uma combinação destes, nos tempos de: 30 minutos, 1, 3 e 10 dias. As avaliações mostraram que a Ang II não altera pressão arterial, sugerindo que os aumentos observados de IL-6 e TGFβ sejam decorrentes de ação direta da Ang II. A Ang II promove aumento tanto agudo de TGFβ, possivelmente associado à ação proteolítica da plasmina e alterações vasculares transitórias compatíveis com aumento de permeabilidade, como crônico de TGFβ, possivelmente associado ao aumento da expressão gênica, levando ao aumento da deposição de colágeno vascular. / This study, evaluated the effect of the intermittent exposure to Angiotensin II (Ang II), taking in account a non-pressor dose on the release of inflammatory cytokines such as interleukin-6 (IL-6), transforming growth factor beta (TGFβ) as well PAI-1 and plasminogen /plasmin. The study was conducted on male mice C57BL/6 subjected to the treatment with angiotensin II (30 ng/kg), losartan (30 mg/kg) or a combination thereof, at times: 30 minutes, 1, 3 and 10 days. The evaluations showed that Ang II did not change blood pressure, suggesting that the increases of IL-6 and TGFβ may be by due to direct action of Ang II. Ang II promotes both acute increase of TGFβ, possibly associated with the proteolytic action of plasmin and transient vascular changes consistent with increased permeability, such as chronic increase of TGFβ, possibly associated with increased gene expression, leading to increased vascular collagen deposition.

O fator de crescimento transformante beta tipo 1, TGF-β1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-β1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-β1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-β1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-β1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-β1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-β1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-β1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-β1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos. / The transforming growth factor beta 1, TGF-β1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-β1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-β1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-β1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-β1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-β1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-β1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-β1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Despite extensive research on an effective treatment for acute renal injury (AKI), the mortality rate still remains high. Moreover, patients who survive AKI are at high risk for chronic progressive kidney disease. Mesenchymal stromal cells derived from human amniotic fluid (hAFSCs) are a new source of stem cells which express renal progenitor markers (CD24). The possibility of combining gene and cell therapy allows stem cells to be manipulated to overexpress vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), two of the most important growth factors for kidney regeneration. Therefore, the aim of this study was to evaluate whether hAFSCs overexpressing VEGF and HGF demonstrate a nephroprotective effect due to their mitogenic and anti-inflammatory effect, leading to a long term inhibition of fibrosis. In the first phase of this study, we isolated hAFSCs from human amniotic fluid samples, characterized their immunophenotypic properties and differentiation capacity and selected a clonal lineage which expresses CD24 and CD117 markers. This lineage was subqequently transduced with lentiviral vectors (LV) encoding VEGF and HGF. In a second phase, renal ischemia and reperfusion (IR) injury was induced in a rat model by clamping the renal pedicle for 50 minutes in 50 male Wistar rats. Treatment groups (n = 10 per group) were assigned as follows: a control group treated with Chang Medium only; a group which received non-transduced AFSC (1x106 cells/animal); a group which received AFSC transduced with LV-VEGF (1x106 cells/animal); a group which received AFSC transduced with LV-HGF (1x106 cells/animal); and a group treated with AFSC transduced both with LV-VEGF (0,5x106 cells/animal) and AFSC transduced with LV-HGF (0,5x106 cells/animal). Serum creatinine was measured at 24 hours, 48 hours and 2 months after IR injury and histological analysis was performed to analyze following parameters: tubular necrosis and hyaline cast formation by PAS and H&E staining at 48 hours, and interstitial fibrosis by Masson s Trichrome and Picrosirius Red staining at 2 months. Additionally, the expression of KI-67, α-SMA and TGF-β1 was assessed by immunohistochemistry. The results showed a beneficial effect of AFSCs delivered to rats with IR injury, which was characterized by a faster improvement in renal function and a lower fibrotic index. However, administration of hAFSCs overexpressing VEGF and HGF resulted in an even better outcome compared to non-transduced AFSCs. As early as 24 hours after AFSC delivery, a nephroprotective effect was observed after both hAFSC and hAFSC VEGF + HGF treatment, which was characterized by significantly lower creatinine values compared to those of the control group. At 48 hours, all treatment groups still demonstrated a significant increase in creatinine values compared to sham animals, except in the hAFSC HGF + VEGF group. In hAFSC HGF + VEGF and hAFSC VEGF treatment groups, a significant increase in renal tubules proliferation was observed, measured by an increase in KI-67 expression, which is probably due to the effect of VEGF overexpression. Furthermore, we observed a decrease in α-SMA and TGF-β expression at 48 hours in the non-transduced hAFSC, hAFSC HGF and hAFSC VEGF + HGF groups. As TGF-β1 is involved in transdifferentiation of tubular epithelial cells to α-SMA-positive myofibroblasts which increases extracellular matrix deposition, the reduction in the expression of α-SMA and TGF-β indicates an inhibition of fibrosis. Although VEGF and HGF have both been described to have nephroprotective properties, we interestingly observed that the hAFSC expressing both VEGF and HGF resulted in more pronounced kidney damage compared to non-treated animals when treated with 1x106 cells/animal, which suggests that toxic side effects are possibly induced by high secretion levels of growth factors. In conclusion, cellular therapy using the combination of hAFSCs transduced with lentiviral vectors encoding VEGF and HGF, resulted in a stronger nephroprotective effect than non-transduced hAFSC delivered to rats with I/R injury, which was characterized by an increased mitosis index, an improved renal function and an inhibition of genes involved in fibrosis resulting in a lower fibrotic index at two months. / O tratamento eficaz para a lesão renal aguda tem melhorado nos últimos anos, sendo objeto de inúmeras pesquisas, no entanto a taxa de mortalidade desta patologia ainda permanece elevada. Além disso, pacientes que sobrevivem após evento isquêmico possuem altos riscos de doença renal crônica progressiva. As células mesenquimais estromais do líquido amniótico humano (hAFSC) são uma nova fonte alternativa de células-tronco que expressam marcadores progenitores renais (CD24). A possibilidade da associação das terapias gênica e celular permite a manipulação dessas para superexpressar o fator de crescimento vascular endotelial (VEGF) e o fator de crescimento de hepatócitos (HGF), dois dos fatores de crescimento mais importantes para a regeneração renal. Diante disso, o objetivo desse estudo foi avaliar se as hAFSCs transduzidas com VEGF e HGF possuem maior ação nefroprotetora, por meio de efeito mitótico e anti-inflamatório a curto prazo, levando a uma inibição da fibrose a longo prazo em modelos de isquemia e reperfusão renal. Na primeira fase do experimento, isolou-se, caracterizou-se as propriedades imunofenotípicas e a capacidade de diferenciação das hAFSCs e após selecionou-se uma linhagem clonal que expressasse os marcadores CD24 e CD117. Após essa linhagem foi transduzida com vetores lentivirais (VL) codificando VEGF e HGF. Na segunda fase, induziu-se lesão de isquemia e reperfusão (IR) renal pelo clampeamento do pedículo renal por 50 minutos, em 50 ratos Wistar, machos. O grupos de tratamento foram divididos como (n=10 por grupo): grupo controle, tratado somente com o meio Chang; grupo tratado com hAFSC não transduzidas (1x106/rato); grupo tratado com hAFSC transduzidas com LV-VEGF (1x106/rato); grupo tratado com hAFSC transduzidas com LV-HGF (1x106/rato) e o grupo tratado tanto com hAFSC transduzidas com LV-VEGF (0,5x106/rato) quanto LV-HGF (0,5x106/rato). A creatinina sérica foi mensurada em 24 horas, 48 horas e 2 meses após a lesão IR e as análises histológicas foram realizadas para avaliar os seguintes parâmetros: necrose tubular e formação de cilíndros hialinos pelas colorações de PAS e H&E em 48 horas e fibrose intersticial pelas colorações Tricrômico de Masson e Picrosirius Red em 2 meses Adicionalmente, a expressão de KI-67, -SMA e TGF- foram analisadas por imunoistoquímica em 48 horas. Os resultados permitiram observar um efeito benéfico da terapia com hAFSCs em lesões de IR pela melhora mais rápida da função renal e menor índice fibrótico a longo prazo, no entanto obteve-se um efeito ainda melhor quando associaram-se as hAFSCs. transduzidas com VEGF e HGF comparado com as hAFSC não transduzidas. Já em 24 h observou-se o efeito renoprotetor nos grupos hAFSC e hAFSC VEGF + HGF pelo valor significativamente menor da creatinina comparado com o controle. Em 48h todos os grupos ainda apresentavam valores significativamente elevados de creatinina comparado com os ratos sham, exceto o grupo hAFSC HGF + VEGF. Observou-se também que os grupos hAFSC VEGF +HGF e hAFSC VEGF tiveram um aumento significativo na proliferação tubular renal, provavelmente pelo efeito da superexpressão de VEGF. Além disso, observou-se redução da expressão de α-SMA e TGF-β em 48h nos grupos hAFSCs não-transduzidas, hAFSC VEGF+HGF e hAFSC HGF. Como o TGF- β1 está envolvido na transdiferenciação de células epiteliais tubulares em miofibroblastos α-SMA-positivos, o qual aumenta a deposição de matriz extracelular, a redução na expressão de α-SMA e TGF-β são indicadoras de inibição da fibrose. Apesar de serem descritos diversos benefícios nefroprotetores do VEGF e do HGF observou-se nesse estudo uma lesão renal mais pronunciada do que o controle nos grupos hAFSC VEGF e hAFSC HGF, tanto em relação à função renal quanto à necrose tubular, o que sugere um efeito tóxico causado pela alta concentração de secreção desses fatores de crescimento. A terapia celular utilizando a combinação de hAFSCs transduzida com vetores lentivirais codificando VEGF e HGF resultou em efeito nefroprotetor ainda maior do que sua forma não transduzida após evento isquêmico renal, o qual foi caracterizado pelo aumento do índice mitogênico, melhor função renal e inibição de genes fibrogênicos, levando a um menor índice fibrótico em dois meses.

L'infiltration des mélanomes par les macrophages (TAM) est souvent corrélée à un mauvais pronostic. Cependant, les mécanismes qui régulent le recrutement et la fonction de ces cellules restent encore mal compris. Des études récentes ont montré un rôle majeur de la sphingosine kinase 1 (SK1) tumorale, l'enzyme qui produit la sphingosine-1-phosphate (S1P), dans le remodelage du stroma associé à la tumeur. Le but de ce projet a été d'étudier le rôle de la SK1 tumorale sur le microenvironnement inflammatoire, et en particulier les macrophages, lors du développement des tumeurs mélaniques. In vitro, nous avons montré que l'inhibition de SK1 dans les cellules de mélanome : 1) bloque la migration des macrophages. A l'inverse, la surexpression de cette kinase dans les cellules tumorales stimule la migration des cellules inflammatoires. Cet effet est dépendant de la S1P et de sa fixation sur les récepteurs S1PR présents à la surface des macrophages ; 2) réduit la sécrétion de TGF-ß et 3) stimule la différenciation des macrophages vers un phénotype M1 antitumoral. Ce phénomène n'est pas dépendant de la S1P, ni des S1PRs, mais de la sécrétion de TGF-ß par les cellules tumorales. En effet, la différenciation macrophagique peut-être réversée par l'addition de TGF-ß recombinant dans le milieu de sécrétion des cellules tumorales. In vivo, nos résultats montrent que la greffe orthotopique, i.e. intradermique, de cellules de mélanome murin invalidées pour la SK1 à des souris syngéniques C57BL/6 est associée à une réduction de la croissance tumorale, comparée à des souris ayant reçu des cellules de mélanome contrôles. De plus, l'invalidation de la SK1 tumorale conduit à une augmentation significative de l'expression de cytokines anti-tumorales ainsi qu'à une polarisation Th1 au sein de la tumeur. Ce phénomène s'accompagne d'une réduction du pourcentage de macrophages M2 CD206+MHCIIlow, et à l'inverse, d'une augmentation du pourcentage de macrophages M1 CD206-MHCIIhigh ainsi que de lymphocytes T CD4+ et CD8+ infiltrés dans la tumeur. Ces résultats suggèrent un rôle clé de la SK1 tumorale dans le recrutement et la polarisation des macrophages dans les mélanomes. Ainsi, l'axe SK1/ TGF-ß pourrait constituer une cible thérapeutique prometteuse dans le contrôle de la croissance de cette tumeur. / Melanoma infiltration by macrophages (TAM) is often correlated with poor prognosis. However, the mechanisms that regulate the recruitment and function of these cells remain poorly understood. Recent studies have shown a major role of tumor sphingosine kinase 1 (SK1), the enzyme that produces sphingosine-1-phosphate (S1P), in tumor stroma remodeling. The aim of this project was to investigate the role of tumor SK1 on the inflammatory microenvironment, particularly macrophages, during the development of melanoma. In vitro, we showed that the inhibition of SK1 in melanoma cells: 1) blocks macrophage migration. Conversely, overexpression of this kinase in tumor cells stimulates the migration of inflammatory cells. This effect is dependent on S1P binding to its receptors (S1PR) on the macrophage surface; 2) reduces the secretion of TGF-ß and 3) stimulates macrophage differentiation towards an antitumor M1 phenotype. The latter phenomenon does not depend on S1P nor S1PRs, but on the secretion of TGF-ß by tumor cells. Indeed, macrophage differentiation can be reversed by adding recombinant TGF-ß in the tumor cell-conditioned medium. In vivo, our results showed that orthotopic injection, i.e. intradermal, of murine melanoma cells invalidated for SK1 in C57BL / 6 syngenic mice was associated with a reduction in tumor growth compared to mice having received control melanoma cells. Furthermore, the invalidation of tumor SK1 leads to a significant increase in the expression of anti-tumor cytokines and a Th1 polarization within the tumor. This phenomenon is accompanied by a reduction in the percentage of CD206+MHCIIlow M2 macrophages, and conversely, an increase in the percentage of M1 macrophages CD206-MHCIIhigh as well as CD4+ and CD8+ cells infiltrated into the tumor. These results suggest a key role of tumor SK1 in the recruitment of macrophages and their polarization in melanoma. Thus, the axis SK1 / TGF-ß could be a promising therapeutic target in controlling the growth of this tumor.

La Bone Morphogenetic Protein-1 (BMP-1) est une métalloprotéase impliquée dans la maturation et l'activation de nombreuses molécules extracellulaires. Parmi celles-ci, on trouve notamment les collagènes fibrillaires, les protéines les plus abondante chez l'Homme, ainsi que les facteurs de croissance de la superfamille du TGF-ß, des protéines pléiotropes. Au travers de ses fonctions, BMP-1 joue un rôle crucial au cours du développement embryonnaire mais également durant les processus physiologiques et pathologiques de remodelage tissulaire (cicatrisation, fibroses, croissance osseuse, cancers...).Le projet présenté dans ce manuscrit a consisté à étudier plusieurs fonctions importantes de BMP-1 au niveau moléculaire et à caractériser les conséquences de ces activités au niveau de plusieurs types cellulaires.Dans un premier temps, un test quantitatif a été mis au point afin de pouvoir étudier en temps réel l'effet de BMP-1 sur les collagènes fibrillaires ainsi que les mécanismes de sa régulation. Par la suite, de nouveaux substrats de BMP-1 ont été mis en évidence, parmi lesquels des co-récepteurs du TGF-ß (Bétaglycan, CD109) ainsi qu'une protéine matricellulaire (TSP-1). L'étude de ces activités a permis de caractériser les multiples voies par lesquelles BMP-1 est capable de réguler l'activité du facteur de croissance TGF-ß.De plus, nous avons mis en évidence que le clivage de ces différents substrats entraine une modulation importante du phénotype de plusieurs lignées cellulaires (HT1080, HEK-293T) avec des effets au niveau de l'adhésion, la prolifération et la migration cellulaire. En conclusion, ce travail révèle que les activités de BMP-1 s'étendent bien au-delà de ce qui est actuellement décrit / Bone Morphogenetic Protein (BMP-1) is a metalloprotease known to be involved in the maturation and activation of several important extracellular proteins, including fibrillar collagens and growth factors of the TGF-beta superfamily. As a consequence, it is essential for embryonic development and tissue remodeling and has been clearly involved in lethal diseases such as fibrosis and cancer.This thesis project focused on the major molecular functions of BMP-1 and their implications for the phenotype of several cell types. First, a quantitative and real-time assay was developed to study the effect of BMP-1 and associated regulatory proteins on fibrillar collagens. Then, new BMP-1 substrates, such as TGF-ß co-receptors (Betaglycan and CD109) and matricellular proteins (TSP-1) were characterized in detail. Especially, we evidenced that these activities played a major role in the regulation of the TGF-ß pathway.Furthermore, we shown that these BMP-1 activities induce major phenotype changes (adhesion, proliferation, migration) in several cell lines including HT1080 and HEK-293T. Altogether, this work reveals that BMP-1 substrates extend far beyond what is presently described and open several perspectives for future studies