O adenocarcinoma pancreático é a neoplasia maligna mais comum do pâncreas. A alta taxa de mortalidade deve-se ao diagnóstico tardio e a alta agressividade do tumor. Freqüentemente observam-se indivíduos com neoplasias de mesmo estadio apresentarem sobrevivência diferente. Isso demonstra a necessidade de incluir mais variáveis na caracterização da doença. O processo de angiogênese é essencial para o crescimento tanto do tumor primário, quanto para o metastático. A medida da densidade intratumoral da microvasculatura (DMV) por imunoistoquímica é o método mais confiável para medir a atividade angiogênica tumoral. A perda da função do gene p53 influencia a resposta à quimio e à radioterapia além de regular a angiogênese. A sobrevivência está inversamente relacionada à positividade do p53 e à DMV em neoplasias de mama, pulmão, ovários, estômago, cólon, laringe e bexiga. No adenocarcinoma pancreático os resultados são controversos. Idealizou-se essa pesquisa retrospectiva analisando-se dados clínicos e os resultados de estudos imunoistoquímicos obtidos de adenocarcinomas de pâncreas ressecados com intenção curativa. Analisou-se dados clínicos, patológicos, re-estadiamento e resultados da DMV e da expressão do gene p53 em 49 pacientes. A densidade média de microvasos foi de 46,2 vasos/mm2 sendo que esse valor foi utilizado para dividir os pacientes em grupos de baixa ou alta densidade de vasos. A coloração para p53 nuclear foi considerada positiva em 20 de 49 pacientes (40,8%). A DMV foi significativamente maior nos pacientes com tumores maiores que 3,0 cm e nos pacientes com ressecções incompletas. A expressão do gene p53 e a DMV, não foram fatores preditivos da sobrevivência pós-operatória. Não foi possível verificar relação entre a expressão do gene p53 e a densidade da microvasculatura tumoral / The prognostic significance of microvessel density and the p53 expression was evaluated. Between 1993 and 2006, 49 patients with pancreatic adenocarcinoma were ressected with curative intention. Specimens were stained immunohistochemically with antibodies anti- p53 anti-CD34. Microvessel density (MVD) was assessed scanning ten areas of the tumoral section and counted at a high power in an adequate area. The MVD ranged from 21,2 to 54,2 vessels/mm2 (mean 46,2 vessels/mm2). Specific nuclear staining for p53 was determined positive in 20 patients (40,8%). The overall median survival was 24,1 months after resection and there was no difference in survival rates according to the MVD and p53 positivity. There was also no relation between the MVD and p53 expression. MVD and p53 expression could not predict survival in these patients with pancreatic adenocarcinoma. There was no correlation with p53 expression and intratumoral microvessel density. High MVD was associated with tumor size grater than 3,0 cm and positive margins

A galectina-3, um membro da família das proteínas de ligação a glicanas, tem sido objeto de intensa pesquisa nos últimos anos devido ao seu importante papel na biologia tumoral, como a proliferação, transformação, apoptose, angiogênese, adesão, invasão e metástase tumoral. As diferentes funções de galectina-3 nas células tumorais resultam das suas diversas localizações inter- e subcelulares que lhe permite interagir com diferentes proteínas. Esta tese teve como objetivo identificar um papel específico de galectina-3 na regulação da via de sinalização Notch, que cada vez mais tem sido associada com a progressão tumoral e angiogênese. Inicialmente, demonstramos que galectina-3 interage com o receptor Notch-1 e modula diferencialmente a ativação da via pelos ligantes DLL4 e Jagged1. A galectin-3 regulou a expressão dos ligantes de Notch assim como o receptor Notch-1 e extracelularmente recuperou a ativação de Notch na ausência de galectina-3 endógena. Em câncer gástrico humano, a galectina-3 encontrou-se positivamente correlacionada com a expressão de Jagged1, enquanto que a galectina-1, um outro membro da família das galectinas, foi positivamente correlacionado com DLL4. De seguida estudou-se o papel biológico da regulação da via Notch pela galectina-3 na angiogênese. Demonstramos que nas células endoteliais, galectina-3 liga e aumenta a meia vida de Jagged1 promovendo a ativação preferencial da Jagged1/Notch em vez de DLL4/Notch de uma forma independente de VEGF. Verificamos que condições de hipóxia alteraram a expressão de galectina-3 assim como o status de glicosilação das células endoteliais de forma a promover a ativação de Jagged1/Notch e o aumento de angiogênese. A superexpressão de Jagged1 num modelo de carcinoma de pulmão de Lewis, acelerou o crescimento tumoral in vivo que foi inibido em camundongos Lgals3-/-. Por fim, avaliou-se o papel de galectina-3 na resistência das células tumorais a quimioterápicos. Observamos que a expressão de sialil-Tn, um produto biossintético da ST6GalNAc-I, diminuiu in vitro como in vivo a presença e os sítios de ligação de galectina-3 na superfície da células levando à sua acumulação no meio intracelular. Extracelularmente, galectina-3 não levou à indução de morte celular, no entanto contribuiu para a morte induzida por quimioterápicos. As células expressando sialil-Tn encontraram-se protegidas. Em amostras de tumor gástrico, os sítios de ligação de galectina-3 encontraram-se negativamente correlacionados com a expressão de sialil-Tn. Este conhecimento possui implicações diretas no desenvolvimento de estratégias visando o controle do crescimento tumoral e angiogênese e abre novas perspectivas no combate à resistência tumoral à terapia / Galectin-3, a member of a family of glycan binding proteins has been the subject of an intense research over the past few years due to its important role in cancer biology, such as cancer cell growth, transformation, apoptosis, angiogenesis, adhesion, invasion and metastasis. The different roles of galectin-3 on cancer cells behavior appears to have originated from its diverse inter- and subcellular localizations where it interacts with several different binding partners. The aim of this thesis was to pinpoint a specific role for galectin-3 in regulating Notch signaling pathway in cancer. Notch signaling has emerged as an important pathway in carcinogenesis, and activated Notch-1 signaling has being associated with cancer progression and angiogenesis. Initially, we found that galectin-3 was able to interact with Notch-1 receptor and to differentially modulate Notch signaling activation by DLL4 and Jagged1 ligands. Galectin-3 was found to regulate the expression of the Notch ligands and Notch-1 receptor and its extracellular form was able to rescue Notch activation in the absence of endogenous galectin-3. In human gastric cancer, galectin-3 was positively correlated with the expression of Jagged1 whereas galectin1, another member of the galectin family, was positively correlated with DLL4. Furthermore, we studied the biological role of Notch regulation by galectin-3 in angiogenesis. We showed that, in endothelial cells, galectin-3 binds to and increases Jagged1 protein half-life promoting Jagged1/Notch over DLL4/Notch signaling in a VEGF independent way. Hypoxic conditions changed galectin-3 expression and the glycosylation status of endothelial cells, acting in concert to promote Jagged1/Notch activation and sprouting angiogenesis. Jagged1 overexpression in Lewis lung carcinoma accelerated tumor growth in vivo that was prevented in Lgals3-/- mice. Finally, we evaluated the role of galectin-3 in cancer cell resistance to therapy. We found that the expression of sialyl-Tn, a biosynthetic product of ST6GalNAc-I, was able to decrease cell surface galectin-3 and galectin-3-binding sites both in vitro and in vivo leading to an intracellular accumulation of this protein. Exogenously added galectin-3 was found to have no effect on cancer cell death but contributed to chemotherapy-induced apoptosis. Sialyl-Tn expressing cells were protected. In human gastric cancer samples, galectin-3 binding sites were negatively correlated with the expression of sialyl-Tn. This knowledge has direct implications for the development of strategies aimed at controlling tumor growth and angiogenesis and open novel perspectives to overcome tumor resistance to therapy

Orientador: Oslei Paes de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-23T09:15:14Z (GMT). No. of bitstreams: 1 ToralRizo_VictorHugo_D.pdf: 3216317 bytes, checksum: f10b9daa311d7ae2e304dc1ea8925abf (MD5) Previous issue date: 2013 / Resumo: O linfoma difuso de grandes células B (LDGCB) é o linfoma da cavidade bucal mais comum. Alguns dos LDGCB podem apresentar células grandes morfologicamente similares às células Hodgkin e Reed/Sternberg (HRS) dos linfomas de Hodgkin clássico (LHC). O objetivo deste estudo foi comparar os LDGCB bucal que apresentem células HRS-like (LDGCB-HRS) com o linfoma de Hodgkin primário nodal, considerando os aspectos histológicos e imunoistoquímicos (IQs), angiogênese, índice de mastócitos e células dendríticas (CD), por meio de um amplo painel IQ. Quize casos foram estudados, nos quais sete eram LDGCB-HRS like e oito eram LHC nodal. Para a análise dos aspectos histológicos e IQs foram utilizados os seguintes anticorpos: CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforina, granzyme B, FASN, Ki-67, LMP-1; e EBER1/2. Já para a análise da angiogênese foram utilizados os anticorpos CD34, CD31, D2-40, CD105, vWF e VEGF; e para o índice de mastócitos utilizou-se o mast cell triptase. Finalmente, para avaliar a expressão IQ das CD os anticorpos CD1a, CD83, CD123, CD207, S-100 e FXIIIa foram utilizados. Todas as lâminas foram escaneadas e as células HRS-like, mastócitos e CD imunopositivas foram analisados, assim como os parâmetros morfométricos da angiogênese. Os resultados mostraram que a imunoexpressão foi postiva em 100% de casos de LHC e em 57% dos casos de LDGCB de boca, enquanto que LCA, CD20 e CD79a foram exclusivos para todos os LDGCB, e apenas CD15 foi exclusivo para os LHC. Angiogênese e o índice de mastócitos estavam aumentados em ambas as lesões, e entre elas, o LHC obteve maiores valores que o LDGCB da cavidade bucal em todos os anticorpos analisados. Por fim, o índice de CDs foram estatisticamente significante entre os grupos, exceto para CD83, que não mostrou nenhuma diferença estatística. A distribuição de CD foi reconhecida principalmente na área tumoral e ao redor das células neoplásicas em ambas as entidades. Foi possível concluir que os LDGCB com células HRS-like da cavidade bucal devem ser incluídos no diagnóstico diferencial de LHC da cavidade bucal. Quando da avaliação destes casos, a analise morfológica detalhada assim como o uso de um amplo painel de IQ são recomendados para realizar o diagnóstico correto. A angiogênese é essencial para o desenvolvimento de LDGCB da cavidade bucal, e quaisquer dos anticorpos CD34, CD31 e vWF podem ser utilizados para avaliar os parâmetros morfométricos. A presença significativa de CD nestes linfomas provavelmente desempenha um papel patologicamente relevante nos linfomas. Nossos resultados sugerem que o aumento no número de CD parece ser um fator contribuinte para a resposta imune estimulada pelo crescimento tumoral / Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common oral lymphoma. Some DLBCLs can present large cells morphologically similar to Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). The objective of this study was to compare oral DLBCL presenting HRS-like cells (DLBCL-HRS like) with primary nodal cHL, considering the following aspects: histological and immunohistochemical (IHC), angiogenesis, index of mast cells and dendritic cells (DCs); through a broad immunohistochemical panel. Fifteen cases were studied, of which, seven were DLBCL-HRS like and eight were nodal cHL. For histological and IHC aspects, immunoexpression of CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforin, granzyme B, FASN, Ki-67, LMP-1; and EBER1/2, were assessed. As for angiogenesis analysis, the antibodies used were CD34, CD31, D2-40, CD105, vWF and VEGF; and for the index of mast cell were used the mast cell tryptase. Finally, for IHC expression of DCs, the antibodies used were CD1a, CD83, CD123, CD207, S-100, and FXIIIa. All slides were scanned and positive immunoreactive cells HRS-like, mast cell and DCs were analyzed, as well as morphometric parameters of angiogenesis. The results showed that the immunoexpression of CD30 was 100% positive in cHL and 57% in oral DLBCL HRS-like, while LCA, CD20 and CD79a were exclusive for all oral DLBCL, and only CD15 was exclusive for cHL. Angiogenesis and mast cell index values were increased in both lesions and between them, cHL was greater than oral DLBCL with all antibodies studied. Finally, DC subsets were statistically significant between groups, except CD83, which did not show statistical significance. The distribution of DCs was mainly in the tumor area, around neoplastic cells in both entities. It was possible to conclude that DLBCL-HRS should be included in the differential diagnosis of oral cHL. When evaluating these cases, a detailed morphologic and a broad IHC analyses for the correct diagnosis are recommended. Angiogenesis is essential to the development of DLBCL of the oral cavity and any of the antibodies CD34, CD31 and vWF could be used to evaluate morphometric parameters. The presence of significantly higher numbers of DCs in these lymphomas could suggest that these cells are likely to play a pathological relevant role in lymphomas. Our findings suggest that increased number of DCs in lymphomas appears to be a factor contributing to the immune response against tumor growth / Doutorado / Patologia / Doutor em Estomatopatologia

A. Background<p><p>Skin wound healing (WH) is a dynamic and extremely determinate process of cellular, humoral and molecular mechanisms which begins directly after wounding and can last for years. WH is described as is an intricate process in which the skin (or another organ-tissue) repairs itself after injury. The process of skin WH occurs through the actions of an interplay of cells, growth factors and cytokines leading to wound closure.<p><p>WH occurs in three precisely and highly programmed phases: the inflammatory phase (day 0 to day 7) followed by the proliferative phase or vasculogenic phase (day 7 to day 21) and finally the remodeling phase (2 days - up to 2 years). For a successful healing, all three phases must occur in the proper sequence and time frame.<p><p>Many factors can interfere with one or more phases of the WH process, thus causing improper or impaired healing. The proliferation phase, in particular, requires the participation of various cells types such as fibroblasts, endothelial cells (ECs) and endothelial progenitor cells (EPCs), to produce a healthy well-vascularized granulation tissue for epithelization and wound closure.<p><p>A.1 Wound Healing And Obesity<p><p>In 2008, over 1.4 billion adults, 20 and older, were overweight. Of these, obesity has been shown to affect over 500 million people (OMS website). Moreover, the prevalence of obesity continues to rise, and by 2018, it is estimated that obesity will cost $ 347 billion annually.<p><p>Each year, in the US, approximately 33 million overweight and obese patients undergo surgery. Obesity causes a number of known health problems and increased post-surgical complications such as wound infection, dehiscence, hematoma and seroma. Surgeons anecdotally report WH complications among obese patients; however, little research has been conducted to investigate the mechanisms mediating impaired obesity-related WH. <p><p>Some previous work on diabetic patients and diabetic mice showed an imbalance between pro-oxydant and anti-oxydant genes as well as impaired EPCs proliferation and tube formation during the WH process. More then a hundred cytologic factors have been found to impair WH in the type 2 diabetic patient. It is a very complex and multifactorial problem involving decreased growth factors secretion, impaired keratinocyte and fibroblast functions, impaired EPs function, alteration of the macrophage function and granulation tissue synthesis, etc. <p><p>Based on these findings and because obesity is associated with the development of type 2 diabetes, we hypothetize that, impaired balance between pro-apoptotic/anti-apoptotic and pro- oxydant /anti-oxydant genes is involved in impaired WH. Furthermore, we hypothetize that impaired EPCs function leads to the perturbation of the proliferation phase of obesity impaired WH.<p><p>A.2. Wound Healing and Age<p><p>The world population is aging; by 2030, nearly 20% of Americans, (± 72 million people), will be 65 years old and older. In 2010, 17% of the European population was over the age of 65. By 2060, it is projected that the share of those aged 65 and over will rise to 30%, accounting for more then 150 million people. (ec.europa.eu) These aging subjects undergo an increasing number of surgical procedures: in the past two decades, the percentage of surgeries in patients over 65 has doubled to nearly 40%.<p>As a corollary, it is well established knowledge that elderly WH is impaired. However, little is known about the underlying mechanisms of age-related impaired WH.<p><p>As previously mentioned, adult BM-derived EPCs contribute to peripheral tissue repair and regeneration. In light of the abundant literature suggesting that neovascularization is impaired in the elderly, we characterize a novel model of senile cutaneous WH and investigate the role that vasculogenesis plays in the pathogenesis of age related impaired WH.<p>Aged mice colonies have traditionally been the model for aged small mammalian research, however, the ability to use a readily-available transgenic mouse model with features of accelerated aging would aid in the exploration of targeted therapies and a great number of age-related investigations.<p><p>We hypothesize that the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24-/-) mouse mimics physiological ageing and can be used as a novel model for the study of senescent WH. We further hypothetized that impaired balance between pro-apoptotic/anti-apoptotic and pro-oxydant /anti-oxydant genes as well as impaired EPCs function are responsible for the impairment of the proliferative phase, leading to overall impaired WH.<p><p>A.3 Aims<p><p>Recently, a great deal of research has been directed at understanding the critical factors inducing poorly healing wounds. However, a lot remains unclear.<p><p>It is now well accepted that new blood vessel formation occurs not only by angiogenesis (blood vessels formation from a preexisting network of capillaries), but also by vasculogenesis (blood vessels formation from BM SCs recruitment) and that EPCs contribute to as much as 25% of new blood vessels formed in healing tissues4. They are mobilized from the BM in response to injury and production of local cytokines, are incorporate into wounds and play an integral role in systemic tissue repair. <p><p>Based on this finding, we hypothesized that co-morbidities related impaired WH may be due, in part, to decreased EPCs number, migration/homing, and/or function resulting in impaired vasculogenesis. Because age and/or obesity have been shown to be one of the most common predictors of altered WH, we decided to focus on these two parameters.<p><p>Following a bedside to bench approach the purpose of this work was to 1) develop coherent and translatable models of co-morbidity digging in the physiologic/pathologic mechanisms underlying altered healing in obese and senile mice; 2) develop targeted therapeutics to improve impaired WH.<p><p>B. Material and Methods<p><p>B.1 Human Model<p><p>Since obesity impairs WH and BM EPCs are important for tissue repair, we hypothesize that obesity- impaired WH is due, in part, to impaired EPCs mobilization, trafficking, and function. Peripheral blood was obtained from non diabetic, obese (BMI > 30, n = 25), and non obese (BMI < 30, n = 17) subjects. Peripheral blood human EPCs were isolated, quantified, and functionally assessed.<p>As for aged impaired WH, EPCs of aged subjects have already been found to have decreased adhesion, migration and proliferative properties as well as being decreased in number in elderly patients undergoing surgery compared to younger patients.<p><p>B.2. Mice Models<p><p>Two models of WH were developed and characterized.<p>In order to isolate the effect of obesity on EPCs and WH, OB non-diabetic female TallyHo/JngJ mouse were selected (Female mice don’t express hyperglycemia and hyperinsulinemia). Female SWR/J non-OB mice were used as control mice. In order to limit variables, TallyHO/JngJ obese mice were selected over other OB mice that exhibit a polygenic type of obesity (Jackson Laboratory Website). By selecting this mouse model, we have excluded in our selection of the ideal model common confounding factors such as hyperglycemia, hyperinsulinemia, immune disorders.<p><p>Zmpste24 is a metalloproteinase involved in the maturation of lamin A (LmnA), an essential component of the nuclear envelope. When Zmpste24 or LmnA are knocked-out, mice exhibit profound nuclear architectural abnormalities and histopathological defects that phenocopy an accelerated aging process. Of crucial importance, the lamin-A dependent nuclear alterations seen in Zmpste24-deficient mice have also been found in human physiological aging. We defined the utilization of the Hutchinson-Gilford Progeria Syndrome (HGPS) Zmpste24 deficient (Zmpste24- /-) mouse as a novel model for the study of senescent WH (controls used were C57BL/6J mice).<p><p>B.3. Wounding Model and Data Collection<p><p>All mice group underwent wounding using a stented wound model developed in our laboratory and previously published. Briefly, paired 6-mm circular, full-thickness wounds extending through the panniculus carnosus were made on the dorsal skin of the mouse. An O-ring, 12-mm splint made of silicone sheeting was then sutured to the skin around the wound. To minimize wound contraction and reliably recapitulated the granulation and re-epithelialization seen in human WH by secondary intention. Time to wound closure was measured using standardized digital photographs taken on days 0, 7, 14, and 21. Wound closure was calculated as a percentage of the original wound.<p><p>For each model, EPCs were harvested, quantified by flow-cytometry and their function tested. Wounds were harvested at various time points and RNA, DNA and protein analysis were conducted. Finally immunohistochemistry to assess epidermal thickness, vascularity and WH were also realized.<p><p>In a second step, after characterization of the models, local (using targeted siRNA gel) and systemic therapies (using AMD3100, a PC mobilizer) were applied on the wounds and compared to controls. WH was monitored. We conducted the previously mentioned analysis (RT-PCR, ELISA and DNA analysis) on the harvested samples.<p><p>All values are expressed as a mean ± standard error of mean (SEM). The number of mice per treatment group was determined using G*Power (G*Power©, Melbourne, Australia) to provide a power greater than 0.80. Student T test was realized to compare two groups among each other.<p><p>C. Results<p><p>C.1. Human EPCs Have Impaired Function<p><p>There was no difference in the number of baseline circulating human EPCs in non-diabetic OB and non-OB<p>subjects, but EPCs from OB subjects had impaired adhesion (p<0.05), migration (p<0.01), and proliferation (p<0.001).<p><p>C.2. Obesity and Wound Healing<p><p>TallyHo/JgnJ OB mice demonstrated significantly impaired healing when compared to SWR/J control mice. They healed at an average of 28 ± 2 days (p<0.05). Post-wounding circulating EPCs were quantified and wounds were analyzed. Circulating EPCs recruitment is impaired in wounded TallyHo/JngJ mice and their wounds shown significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Their wounds are characterized by increased apoptosis, increased DNA damage and impaired pro-/anti-oxydant balance. Immunonistochemistry and histology showed decreased vascular vessels in TallyHo/JngJ wounds and thinner epidermal thickness.<p><p>In the local treatment phase, local p53 silencing consistently improved WH to a nearly normal healing time (wounds healed in 18 ± 2 days, p<0.05). sip53 treatment showed a significant decrease in pro-apoptotic markers (p53, Bax, PUMA p<0.05) and a significant increase in angiogenic markers (VEGF, SDF-1α, HIF-1α) with increased blood vessel formation and decreased DNA damage.<p><p>C.3. Age and Wound Healing<p><p>In these experiments, we show that not only is Zmpste24-/- WH impaired when compared to C57BL/6J mice (Zmpste24-/- mice healed at average 40 days ± 2 days p<0.05) at all time points but that they also showed decreased vascularity and proliferation in the wound bed (p<0.05).<p><p>Histological analysis was performed utilizing hematoxylin and eosin staining to assess epidermal thickness, CD31 immunofluorescence to assess vascular density, p53 and caspase 3 to assess apoptosis, 8’OHdG staining to assess DNA damage and PCNA to assess proliferation. Epidermal thickness was significantly decreased in Zmpste24-/- animals compared to WT as well as vascular density, and proliferation in Zmpste24-/- wound tissue (p<0.05). <p><p>Circulating vasculogenic EPCs recruitment was impaired in Zmpste24-/- mice and their wounds showed significantly decreased new blood vessel formation through decreased HIF-1α/SDF-1α signaling (p<0.05). Zmpste24-/- wounds are characterized by increased apoptosis and an abnormal rise in ROS.<p>In the treatment phase, local p53 silencing consistently improved healing by more then a two fold (18 ± 2 days). VEGF production was significantly increased and pro-apoptotic factors were significantly downregulated in siRNA-treated Zmpste24-/- mice (p<0.05). DNA damage due to ROS production was also shown to be significantly decreased following treatment. Our results suggest a vasculogenic dysfunction in wound closure and showed that the specific knock down of p53 significantly improves WH.<p><p>Because EPCs showed impaired function, lower peripheric blood counts and impaired SDF-1α/HIF-1α signaling, we hypothesized that improving their mobilization by using a progenitor cell mobilizer, AMD3100, known to mobilize SCs from the BM, in a systemic treatment phase will improve WH. Peripheral blood counts were significantly increased and time to wound closure significantly decreased (20 days ± 2, p<0.05). Vasculogenic markers and anti- apoptotic molecules were upregulated compare to non-treated animals.<p><p>D. Conclusions<p><p>Obesity impaired wound closure is a complex problem with many contributory factors. Our results suggest that obesity impairs the BM-derived EPCs response to peripheral injury and this, in turn, impairs wound closure. This impairment is associated with decreased new blood vessel formation and increased DNA damage leading to an increase in the p53 pathway. We also demonstrate that targeted siRNA therapy can partially rescue impaired WH due to obesity. Based on these results we support the encouraging argument that, WH and closure has the potential be improved through specific local and systemic therapies in vivo in our rodent model and that further studies are needed to support this in a clinical environment.<p><p>Impaired WH due to ageing is a complex phenomenon that is partially understood. We demonstrate that the Zmpste24-/- transgenic knockout mouse provides a model for age-related WH investigation. Zmpste24-/- animals heals their wounds with significant delays, showed impaired EPCs mobilization following wounding through an impaired HIF-1α/SDF-1α pathway and increased apoptosis. Furthermore, WH can be improved through specific local siRNA therapy and systemic stem cell mobilization therapies.<p><p>Our results suggest strong similar patterns between obesity and ageing in the way they mediate WH impairments trough (premature) ageing. Our encouraging endeavor to bring WH back to baseline in these diseased models underlines the possibility to reverse the microenvironment alterations and improves EPCs contribution to the WH process. Because EPCs are involved in virtually every tissue repair process happening in the human body, we hope that this work will lead the way for new research in various fields in medicine to improve wound care and quality of life of patients. / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Notre travail a permis tout d’abord d’investiguer les effets des nucléotides extracellulaires sur les cellules<p>dendritiques (DCs) qui sont des cellules présentatrices d’antigènes capables d’initier et de réguler la<p>réponse immunitaire. Afin d’avoir une vue globale de l’action des nucléotides extracellulaires sur les DCs,<p>un profil d’expression génique de l’ATPgS – dérivé stable de l’ATP - a été réalisé par microarray dans les<p>cellules dendritiques dérivées de monocytes (MoDCs).<p>Notre groupe a préalablement montré que malgré que l’ATP est considéré comme un signal de danger, il<p>confère des propriétés immunosuppressives aux DCs (Marteau et al, 2005). Nous nous sommes focalisés<p>sur des régulations géniques pouvant être mises en relation avec un action anti-inflammatoire de l’ATP.<p>Nous avons ainsi démontré que l’ATP était capable d’inhiber la sécrétion des chimiokines MCP-1 et MIP-<p>1a initiée par l’action du LPS, ce qui a pour conséquence de diminuer la capacité des DCs à recruter des<p>monocytes ou d’autres DCs. Ce travail a fait l’objet d’une publication en tant que premier auteur<p>(Horckmans et al, 2005).<p>Un grand nombre d’autres gènes régulés liés à la réponse immune et à l’inflammation a été identifiée<p>dans le profil microarray de l’ATPgS. Nous avions notamment pu identifier une augmentation de la<p>sécrétion de VEGF-A en réponse à l’ATP, amplifiée en présence de LPS. Cette régulation est extrêmement<p>intéressante au vu de l’action immunosuppressive du VEGF sur les DCs. Par ailleurs, cette régulation<p>pourrait constituer un lien entre les DCs et l’angiogénese. Ce travail a fait l’objet d’une publication en tant<p>que premier co-auteur dans la revue Journal of Immunology (Bles et al, 2007).<p>En conclusion, nos données nous ont ainsi permis de montrer que les nucléotides adényliques peuvent<p>avoir par leur action sur les cellules dendritiques une action anti-inflammatoire voire pro-angiogénique,<p>en inhibant le recrutement leucocytaire et une action immunosuppressive en stimulant la sécrétion de<p>VEGF.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Large doses of recombinant growth factors formulated in solution form directly injected into the body is usual clinical practice in treating second-degree scald injuries, with promising results, but this approach creates side effects; furthermore, it may not allow appropriate levels of the factor to be sensed by the target injured tissue/organ in the specific time frame, owing to complications arising from regeneration. In this research, two delivery methods (infusion pumping and local topical application) were applied to deliver recombinant human erythropoietin (rHuEPO) for skin regeneration. First, rHuEPO was given in deep second-degree scald injury sites in mice by infusion pump. Vascularization was remarkably higher in the rHuEPO pumping group than in controls. Second, local topical application of rHuEPO gel was given in deep second-degree scald injury sites in rats. Histological analysis showed that epithelialization rate was significantly higher in the rHuEPO gel-treated group than in controls. Immunohistochemical studies showed that the rHuEPO gel-treated group showed remarkably higher expression of skin regeneration makers than the control group. An accurate method for visualization and quantification of blood vessel networks in target areas has still not been developed up to this point, because of technical difficulties in detecting such thin blood vessels. A method which utilizes a series of steps to enhance the image, removes noise from image background, and tracks the vessels edges for vessel segmentation and quantification has been used in this study. Using image analysis methods, we were able to detect the microvascular networks of newly formed blood vessels (less than 500 μm thickness), which participate in the healing process, providing not only nutrition and oxygen to grow tissues but also necessary growth factors to grow tissue cells for complete skin regeneration. The rHuEPO-treated group showed higher expression of stem cell markers (CD 31, CD 90, CD 71, and nestin), which actively contribute to in-wound-healing processes for new hair follicle generation as well as skin regeneration. Collectively, both rHuEPO group pumping into the systemic circulation system, and injection into the local injury area, prompted mice and rats to form new blood vessel networks in scald injury sites, which significantly participate in the scald healing process. These results may lead to the development of novel treatments for scald wounds.

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Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC), which comprises 85% of pancreatic cancers, is the 4th leading cause of cancer death in the United States with a 5-year survival rate of 8%. While human PDACs (hPDACs) are hypovascular, they also overexpress a number of angiogenic growth factors and receptors. Additionally, the use of anti-angiogenic agents in murine models of PDAC leads to reduced tumor volume, tumor spread, and microvessel density (MVD), and improved survival. Nonetheless, clinical trials using anti-angiogenic therapy have been overwhelmingly unsuccessful in hPDAC. On the other hand, pancreatic neuroendocrine tumors (PNETs) account for only 2% of pancreatic tumors, yet they are very vascular and classically angiogenic, respond to anti-angiogenic therapy, and confer a better prognosis than PDAC even in the metastatic setting. In an effort to compare and contrast the angiogenic transcriptomes of these two tumor types, we analyzed RNA-Sequencing (RNA-Seq) data from The Cancer Genome Atlas (TCGA) and found that a pro-angiogenic gene signature is present in 35% of PDACs and that it is mostly distinct from the angiogenic signature present in PNETs. The pro-angiogenic PDAC subgroup also exhibits a transcriptome that reflects active TGF-β signaling, less frequent SMAD4 inactivation than PDACs without the signature, and up-regulation of several pro-inflammatory genes, including members of JAK signaling pathways. Consequently, targeting the TGF-β receptor type-1 kinase with SB505124 and JAK1/2 with ruxolitinib blocks proliferative crosstalk between human pancreatic cancer cells (PCCs) and human endothelial cells (ECs). Additionally, treatment of the KRC (oncogenic Kras, homozygous deletion of Rb1) and KPC (oncogenic Kras, mutated Trp53) genetically engineered PDAC mouse models with ruxolitinib suppresses murine PDAC (mPDAC) progression only in the KRC model, which shows superior enrichment and differential expression of the human pro-angiogenic gene signature as compared to KPC tumors. These findings suggest that targeting both TGF-β and JAK signaling in the 35% of PDAC patients whose cancers exhibit an pro-angiogenic gene signature should be explored in a clinical trial.

Macular neovascularization type 3, formerly known as retinal angiomatous proliferation (RAP), is a hallmark of age-related macular degeneration and is associated with an accumulation of myeloid cells, such as microglia (MG) and infiltrating blood-derived macrophages (MAC). However, the contribution of MG and MAC to the myeloid cell pool at RAP sites and their exact functions remain unknown. In this study, we combined a microglia-specific reporter mouse line with a mouse model for RAP to identify the contribution of MG and MAC to myeloid cell accumulation at RAP and determined the transcriptional profile of MG using RNA sequencing. We found that MG are the most abundant myeloid cell population around RAP, whereas MAC are rarely, if ever, associated with late stages of RAP. RNA sequencing of RAP-associated MG showed that differentially expressed genes mainly contribute to immune-associated processes, including chemotaxis and migration in early RAP and proliferative capacity in late RAP, which was confirmed by immunohistochemistry. Interestingly, MG upregulated only a few angiomodulatory factors, suggesting a rather low angiogenic potential. In summary, we showed that MG are the dominant myeloid cell population at RAP sites. Moreover, MG significantly altered their transcriptional profile during RAP formation, activating immune-associated processes and exhibiting enhanced proliferation, however, without showing substantial upregulation of angiomodulatory factors.

Immunosenescence is considered a possible factor in the development of age-related macular degeneration and choroidal neovascularization (CNV). However, age-related changes of myeloid cells (MCs), such as microglia and macrophages, in the healthy retina or during CNV formation are illdefined. In this study, Cx3cr1-positive MCs were isolated by fluorescence-activated cell sorting from six-week (young) and two-year-old (old) Cx3cr1GFP/+ mice, both during physiological aging and laser-induced CNV development. High-throughput RNA-sequencing was performed to define the age-dependent transcriptional differences in MCs during physiological aging and CNV development, complemented by immunohistochemical characterization and the quantification of MCs, as well as CNV size measurements. These analyses revealed that myeloid cells change their transcriptional profile during both aging and CNV development. In the steady state, senescent MCs demonstrated an upregulation of factors contributing to cell proliferation and chemotaxis, such as Cxcl13 and Cxcl14, as well as the downregulation of microglial signature genes. During CNV formation, aged myeloid cells revealed a significant upregulation of angiogenic factors such as Arg1 and Lrg1 concomitant with significantly enlarged CNV and an increased accumulation of MCs in aged mice in comparison to young mice. Future studies need to clarify whether this observation is an epiphenomenon or a causal relationship to determine the role of immunosenescence in CNV formation.