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Tumors are composed not only of malignant cells, but also of various types of normal cells, including vascular cells and infiltrating immune cells, which drive tumor development and progression. The tumor vasculature is abnormal and dysfunctional due to sustained tumor angiogenesis driven by high levels of pro-angiogenic factors. Proteins differentially expressed in tumor vessels affect vascular function and the tumor microenvironment and may serve as targets for therapy. The tumor is also a site of sustained chronic inflammation. The recruitment and activation of inflammatory cells significantly influence tumor progression and regression. Targeting molecules regulating tumor angiogenesis and inflammation in the tumor microenvironment is therefore a promising strategy for the treatment of cancer. This thesis is aiming to understand and investigate the molecular regulation of these two processes in tumors. αB-crystallin is a heat shock protein previously proposed as a target for cancer therapy due to its role in increasing survival of tumor cells and enhancing tumor angiogenesis. In this thesis, we demonstrate a novel role of αB-crystallin in limiting expansion of CD11b+Gr1+ immature myeloid cells in pathological conditions, including tumor development. In addition, we show that αB-crystallin regulates leukocyte recruitment by promoting expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin during TNF-α-induced endothelial activation. Therefore, targeting of αB-crystallin may influence tumor inflammation by regulating immature myeloid cell expansion and leukocyte recruitment. Abnormal, dysfunctional vessels are characteristic of glioblastomas, which are aggressive malignant brain tumors. We have identified the orphan G-protein coupled receptor ELTD1 as highly expressed in glioblastoma vessel and investigated its role in tumor angiogenesis. Interestingly, deficiency of ELTD1 was associated with increased growth of orthotopic GL261 glioma and T241 fibrosarcoma, but did not affect vessel density in any model. Further investigation is warranted to evaluate whether ELTD1 serves a suitable vascular target for glioblastoma treatment. Anti-angiogenic drugs targeting VEGF signaling is widely used in the clinic for various types of cancer. However, the influences of anti-angiogenic treatment on tumor inflammation have not been thoroughly investigated. We demonstrate that VEGF inhibits TNF-α-induced endothelial activation by repressing NF-κB activation and expression of chemokines involved in T-cell recruitment. Sunitinib, a small molecule kinase inhibitor targeting VEGF/VEGFR2 signaling increased expression of chemokines CXCL10, CXCL11, and enhanced T-lymphocyte infiltration into tumors. Our study suggests that anti-angiogenic therapy may improve immunotherapy by enhancing endothelial activation and facilitating immune cell infiltration into tumors.

Monocyte recruitment promotes the accumulation of myeloid foam cells in early atherosclerotic plaques. However, initial foam cells form prior to increased monocyte recruitment in hypercholesterolemic Ldlr-/- mice. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alphaX (CD11c). The goal of this thesis was to assess the role of initial foam cells in atherogenesis. The approach was to delete these cells by diphtheria toxin-induced apoptosis in Ldlr-/- bone marrow chimeras. Depletion of CD11c+ leukocytes resulted in significant reductions of intimal lipid accumulation, monocyte recruitment, intimal chemokine expression, but not endothelial cell adhesion molecule expression, at 10 and 21 days of hypercholesterolemia. These data suggest that lipid uptake by resident intimal CD11c-expressing myeloid cells during the earliest stages of atherosclerosis promotes chemokine production that is required for increased monocyte recruitment.

Monocyte recruitment promotes the accumulation of myeloid foam cells in early atherosclerotic plaques. However, initial foam cells form prior to increased monocyte recruitment in hypercholesterolemic Ldlr-/- mice. These initial foam cells are derived from myeloid cells residing in the normal intima, and express integrin alphaX (CD11c). The goal of this thesis was to assess the role of initial foam cells in atherogenesis. The approach was to delete these cells by diphtheria toxin-induced apoptosis in Ldlr-/- bone marrow chimeras. Depletion of CD11c+ leukocytes resulted in significant reductions of intimal lipid accumulation, monocyte recruitment, intimal chemokine expression, but not endothelial cell adhesion molecule expression, at 10 and 21 days of hypercholesterolemia. These data suggest that lipid uptake by resident intimal CD11c-expressing myeloid cells during the earliest stages of atherosclerosis promotes chemokine production that is required for increased monocyte recruitment.

Methylglyoxal (MG) is a reactive dicarbonyl metabolite formed during glucose, protein and fatty acid metabolism. In hyperglycemic conditions, an increased MG level has been linked to the development of diabetes and the accompanying vascular inflammation encountered at both macro- and microvascular levels. The present study explores the mechanisms of MG-induced leukocyte recruitment in mouse cremasteric microvasculature. Biochemical and intravital microscopy studies performed suggest that administration of MG (25 and 50 mg/kg) to mouse cremaster muscle tissue induces dose-dependent leukocyte recruitment in cremasteric vasculature with 84-92% recruited cells being neutrophils. MG treatment up-regulated the expression of endothelial cell (EC) adhesion molecules P-selectin, E-selectin and intercellular adhesion molecule-1 (ICAM-1) via the activation of nuclear factor-κB (NF-κB) signalling pathway and contributed to the increased leukocyte rolling flux, reduced leukocyte rolling velocity, and increased leukocyte adhesion, respectively. The inhibition of NF-κB blunted MG-induced endothelial adhesion molecule expression and thus attenuated leukocyte recruitment. Further study of signalling pathways revealed that MG induced Akt-regulated transient glycogen synthase kinase 3 (GSK3) activation in ECs, which was responsible for NF-κB activation at early time-points (< 1 h). After MG activation for 1 h, the endothelial GSK3 activity was decreased due to the up-regulation of serum- and glucocorticoid-regulated kinase 1 (SGK1), which was responsible for maintaining NF-κB activity at later time-points. Silencing GSK3 or SGK1 attenuated P-selectin, E-selectin and ICAM-1 expression in ECs, and abated MG-induced leukocyte recruitment. SGK1 also promoted cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) activity which was partially involved in ICAM-1 expression. Silencing CREB blunted ICAM-1 expression while P-selectin and E-selectin levels remained unaffected. MG also induced GSK3 activation in isolated neutrophils after 30 min treatment, an effect that was not responsible for MG-elicited Mac-1 expression. These data suggest the sequential activation of GSK3 and SGK1 in ECs as the pivotal signalling mechanism in MG-elicited leukocyte recruitment. Additionally, MG-treatment led to uncoupling of endothelial nitric oxide synthase (eNOS) following MG-induced superoxide generation in ECs. MG triggered eNOS uncoupling and hypophosphorylation associated with superoxide generation and biopterin depletion in EA.hy926 ECs. In cremaster muscle, as well as in cultured murine and human primary ECs, MG increased eNOS monomerization and decreased 5,6,7,8-tetrahydroboipterin (BH4)/total biopterin ratio, effects that were significantly mitigated by supplementation of BH4 or its precursor sepiapterin but not by NG-nitro-L-arginine methyl ester (L-NAME) or 5,6,7,8-tetrahydroneopterin (NH4). These observations confirm that MG administration triggers eNOS uncoupling. In murine cremaster muscle, MG triggered the reduction of leukocyte rolling velocity and the increases in rolling flux, adhesion, emigration and microvascular permeability. MG-induced leukocyte recruitment was significantly attenuated by supplementation of BH4 or sepiapterin or suppression of superoxide by L-NAME confirming the role of eNOS uncoupling in MG-elicited leukocyte recruitment. MG treatment further decreased the expression of guanosine triphosphate cyclohydrolase I in murine primary ECs, suggesting the impaired BH4 biosynthesis caused by MG. Taken together, these data suggest that vascular inflammation and endothelial dysfunction occurring in diabetes may be linked to GSK3/SGK1 regulated adhesion molecule expression, as well as the uncoupling of eNOS evoked by elevated levels of MG. These findings not only provide a better understanding of the role of MG in the development of diabetic vascular inflammation, but also suggest the potential therapeutic targets for MG-sensitive endothelial dysfunction in diabetes.

Resultados preliminares do nosso grupo de pesquisa demonstraram que a inibição da síntese de NO por período de tempo prolongado reduz o recrutamento de leucócitos para focos inflamatórios, dependente, pelo menos em parte, da inibição da interação leucócito-endotélio e da expressão de L-selectina. 0 presente trabalho visou complementar os estudos sobre os mecanismos envolvidos nesta ação antiinflamatória. Para tanto, ratos Wistar machos (180 a 220g) receberam L-NAME (20mg/Kg; v.o.; 14 dias) ou água pela mesma via e período de tempo. Foi avaliada a atividade das enzimas óxido nítrico sintases (NOS) no tecido cerebral por radioimunoensaio; o recrutamento leucocitário para cavidade peritoneal induzido pelo LPS (5mg/kg, 4 horas); as expressões de moléculas de adesão em leucócitos do sangue circulante, no músculo cremaster e nos sinusóides hepáticos por ensaios de citometria de fluxo ou imunohistoquímica; as expressões gênicas de moleculas de adesão, quantificadas por PCR e a secreção/produção de mediadores inflamatórios em leucócitos por ensaios imunoenzimáticos, reacao de Griess ou quimiluminescência. Os resultados obtidos mostraram que: 1) o tratamento com L-NAME reduziu em torno de 90% a atividade das NOS dependentes de Ca+2 em condições basais ou após estimulação in vivo com LPS; 2) as concentrações de NO no plasma e no peritônio inflamado estavam reduzidos em animais tratados com L-NAME (30% vs. controles); 3) a migração de leucócitos polimorfonucleares (PMN) para o peritônio inflamado estava reduzida em animais tratados com L-NAME (40% vs. controles); 4) as expressões de L-selectina e PECAM-1 em leucócitos circulantes; de PECAM-1 no endotélio do músculo cremaster e de VAP-1 no endotélio dos sinusóides hepáticos e músculo cremaster de animais tratados com L-NAME estavam reduzidas; 5) a redução na expressão de L-selectina foi dependente de inibição de sua síntese; 6) a concentração de IL-10 estava major no soro de animais tratados com L-NAME em relação aos controles; 7) a maior concentração de IL-10 circulante pode refletir a produção desta citocina por leucócitos na circulação, uma vez que a concentração de IL-10 também estava maior no sobrenadante de leucócitos circulantes de animais tratados com L-NAME; 8) concentrações reduzidas de IL-1&#946; e LTB4 foram detectadas nos sobrenadantes de neutrófilos obtidos de animais tratados com L-NAME; 9) macrófagos obtidos de animais tratados com L-NAME produziram maiores concentrações de IL-1&#946;, TNF-&#945; e IL-6, e menores concentrações de IL-10 na ausência de estimulação; na vigência estimulação in vitro com LPS os macrófagos de animais tratados com L-NAME produziram menores concentrações de NO. Em conjunto, os resultados obtidos neste trabalho mostram que o tratamento com L-NAME por período prolongado de tempo inibe a atividade das NOS dependentes de Ca+2 e nesta condição reduz as concentrações de NO circulante e no foco da inflamação, além de inibir a migração de leucócitos para o foco inflamatório, confirmando propriedades pro-inflamatórias do NO. Os mecanismos envolvidos na inibição da migração celular parecem compreender a modulação da expressão e/ou síntese das moléculas de adesão constitutivas expressas em leucócitos e no endotélio, além da modulação da secreção de mediadores pró ou antiinflamatórios em leucócitos circulantes e neutrófilos. Por outro lado, os efeitos do tratamento com L-NAME sobre a secreção de mediadores químicos por macrófagos induzem a secreção destes e corroboram a dualidade dos efeitos do NO no processo de recrutamento celular. / Our previous results have demonstrated that in vivo chronic inhibition of nitric oxide synthesis reduces leukocyte recruitment into inflammatory focus, dependent, at least in part, on impaired leukocyte-endothelial interactions and expression of L-selectin. This study aimed to clarify the mechanisms involved in the reduced leukocyte migration observed in L-NAME-treated rats. For this purpose, male Wistar rats (180-220g) were treated with L-NAME (20 mg/kg, oral route, 14 days, dissolved in drinking water); controls animals received water by the same route and period of time. The effectiveness of L-NAME treatment was investigated by examining the activity of nitric oxide synthases (NOS) in the brain tissue using radioimmunoassay. In addition, effects of L-NAME treatment were evaluated in LPS-induced leukocyte recruitment into peritoneal cavity (5mg/kg, 4 hours); expression of adhesion molecules was determined in circulating leukocytes, cremaster muscle and liver sinusoids by flow cytometry and immunohistochemistry assay; gene expressions of adhesion molecules were quantified by PCR and leukocyte secretion of inflammatory mediators was measured by immunoenzimatics assays, Griess reaction or chemiluminescence. Our results show that: 1) L-NAME treatment reduced, around 90%, Ca+2 - dependent NOS activity in the presence or not of in vivo inflammation; 2) concentrations of NO in the plasma and into inflamed peritoneum were reduced in L-NAME-treated animals (30% vs. control animals); 3) migration of PMN leukocytes into inflammed peritoneum was impaired in L-NAME-treated rats (40% vs. control animals); 4) expressions of L-selectin and PECAM-1 in circulating leukocytes, PECAM-1 in endothelium from cremaster muscle and VAP-1 in endothelium from liver sinusoids and cremaster muscle were reduced in L-NAME-treated rats; 5) decrease in L-selectin expression was dependent on inhibition of its synthesis; 6) concentrations of IL-10 was higher in serum from L-NAME-treated rats in comparison to control rats; 7) these higher concentrations of circulating IL-10 can reflect the production of this cytokine by leukocytes from circulation, as IL-10 levels was greater in the supernatant of circulating leukocytes obtained from L-NAME-treated animals; 8) L-NAME treatment disturbed neutrophils ability to secrete IL-1&#946; e LTB4, since these concentrations were lower in the supernatants of neutrophils from L-NAME-treated animals; 9) in absence of stimulation, macrophages obtained from L-NAME-treated rats produced higher concentrations of IL-1&#946;, TNF-&#945; e IL-6, and lower concentrations of IL-10, whereas in presence of in vitro LPS these cells produced lower concentrations of NO. Taken together, our results show that L-NAME treatment administrated for a prolonged period of time inhibits Ca+2 -dependent NOS activity, and in this condition, reduces concentrations of NO in plasma and into inflammatory focus and decreases leukocyte migration to the inflammatory focus thus confirming pro-inflammatory properties of NO. The mechanisms involved in impaired cellular migration seem to involve the modulation of expression and/or synthesis of constitutive adhesion molecules in leukocytes and endothelium, and to interfere in the secretion of pro or anti inflammatory mediators. On the other hand, actions of NO in secreation of chemical mediators by macrophages induces the production of inflammatory mediators and support the duality of NO in the cellular recruitment process.

Resultados preliminares do nosso grupo de pesquisa demonstraram que a inibição da síntese de NO por período de tempo prolongado reduz o recrutamento de leucócitos para focos inflamatórios, dependente, pelo menos em parte, da inibição da interação leucócito-endotélio e da expressão de L-selectina. 0 presente trabalho visou complementar os estudos sobre os mecanismos envolvidos nesta ação antiinflamatória. Para tanto, ratos Wistar machos (180 a 220g) receberam L-NAME (20mg/Kg; v.o.; 14 dias) ou água pela mesma via e período de tempo. Foi avaliada a atividade das enzimas óxido nítrico sintases (NOS) no tecido cerebral por radioimunoensaio; o recrutamento leucocitário para cavidade peritoneal induzido pelo LPS (5mg/kg, 4 horas); as expressões de moléculas de adesão em leucócitos do sangue circulante, no músculo cremaster e nos sinusóides hepáticos por ensaios de citometria de fluxo ou imunohistoquímica; as expressões gênicas de moleculas de adesão, quantificadas por PCR e a secreção/produção de mediadores inflamatórios em leucócitos por ensaios imunoenzimáticos, reacao de Griess ou quimiluminescência. Os resultados obtidos mostraram que: 1) o tratamento com L-NAME reduziu em torno de 90% a atividade das NOS dependentes de Ca+2 em condições basais ou após estimulação in vivo com LPS; 2) as concentrações de NO no plasma e no peritônio inflamado estavam reduzidos em animais tratados com L-NAME (30% vs. controles); 3) a migração de leucócitos polimorfonucleares (PMN) para o peritônio inflamado estava reduzida em animais tratados com L-NAME (40% vs. controles); 4) as expressões de L-selectina e PECAM-1 em leucócitos circulantes; de PECAM-1 no endotélio do músculo cremaster e de VAP-1 no endotélio dos sinusóides hepáticos e músculo cremaster de animais tratados com L-NAME estavam reduzidas; 5) a redução na expressão de L-selectina foi dependente de inibição de sua síntese; 6) a concentração de IL-10 estava major no soro de animais tratados com L-NAME em relação aos controles; 7) a maior concentração de IL-10 circulante pode refletir a produção desta citocina por leucócitos na circulação, uma vez que a concentração de IL-10 também estava maior no sobrenadante de leucócitos circulantes de animais tratados com L-NAME; 8) concentrações reduzidas de IL-1&#946; e LTB4 foram detectadas nos sobrenadantes de neutrófilos obtidos de animais tratados com L-NAME; 9) macrófagos obtidos de animais tratados com L-NAME produziram maiores concentrações de IL-1&#946;, TNF-&#945; e IL-6, e menores concentrações de IL-10 na ausência de estimulação; na vigência estimulação in vitro com LPS os macrófagos de animais tratados com L-NAME produziram menores concentrações de NO. Em conjunto, os resultados obtidos neste trabalho mostram que o tratamento com L-NAME por período prolongado de tempo inibe a atividade das NOS dependentes de Ca+2 e nesta condição reduz as concentrações de NO circulante e no foco da inflamação, além de inibir a migração de leucócitos para o foco inflamatório, confirmando propriedades pro-inflamatórias do NO. Os mecanismos envolvidos na inibição da migração celular parecem compreender a modulação da expressão e/ou síntese das moléculas de adesão constitutivas expressas em leucócitos e no endotélio, além da modulação da secreção de mediadores pró ou antiinflamatórios em leucócitos circulantes e neutrófilos. Por outro lado, os efeitos do tratamento com L-NAME sobre a secreção de mediadores químicos por macrófagos induzem a secreção destes e corroboram a dualidade dos efeitos do NO no processo de recrutamento celular. / Our previous results have demonstrated that in vivo chronic inhibition of nitric oxide synthesis reduces leukocyte recruitment into inflammatory focus, dependent, at least in part, on impaired leukocyte-endothelial interactions and expression of L-selectin. This study aimed to clarify the mechanisms involved in the reduced leukocyte migration observed in L-NAME-treated rats. For this purpose, male Wistar rats (180-220g) were treated with L-NAME (20 mg/kg, oral route, 14 days, dissolved in drinking water); controls animals received water by the same route and period of time. The effectiveness of L-NAME treatment was investigated by examining the activity of nitric oxide synthases (NOS) in the brain tissue using radioimmunoassay. In addition, effects of L-NAME treatment were evaluated in LPS-induced leukocyte recruitment into peritoneal cavity (5mg/kg, 4 hours); expression of adhesion molecules was determined in circulating leukocytes, cremaster muscle and liver sinusoids by flow cytometry and immunohistochemistry assay; gene expressions of adhesion molecules were quantified by PCR and leukocyte secretion of inflammatory mediators was measured by immunoenzimatics assays, Griess reaction or chemiluminescence. Our results show that: 1) L-NAME treatment reduced, around 90%, Ca+2 - dependent NOS activity in the presence or not of in vivo inflammation; 2) concentrations of NO in the plasma and into inflamed peritoneum were reduced in L-NAME-treated animals (30% vs. control animals); 3) migration of PMN leukocytes into inflammed peritoneum was impaired in L-NAME-treated rats (40% vs. control animals); 4) expressions of L-selectin and PECAM-1 in circulating leukocytes, PECAM-1 in endothelium from cremaster muscle and VAP-1 in endothelium from liver sinusoids and cremaster muscle were reduced in L-NAME-treated rats; 5) decrease in L-selectin expression was dependent on inhibition of its synthesis; 6) concentrations of IL-10 was higher in serum from L-NAME-treated rats in comparison to control rats; 7) these higher concentrations of circulating IL-10 can reflect the production of this cytokine by leukocytes from circulation, as IL-10 levels was greater in the supernatant of circulating leukocytes obtained from L-NAME-treated animals; 8) L-NAME treatment disturbed neutrophils ability to secrete IL-1&#946; e LTB4, since these concentrations were lower in the supernatants of neutrophils from L-NAME-treated animals; 9) in absence of stimulation, macrophages obtained from L-NAME-treated rats produced higher concentrations of IL-1&#946;, TNF-&#945; e IL-6, and lower concentrations of IL-10, whereas in presence of in vitro LPS these cells produced lower concentrations of NO. Taken together, our results show that L-NAME treatment administrated for a prolonged period of time inhibits Ca+2 -dependent NOS activity, and in this condition, reduces concentrations of NO in plasma and into inflammatory focus and decreases leukocyte migration to the inflammatory focus thus confirming pro-inflammatory properties of NO. The mechanisms involved in impaired cellular migration seem to involve the modulation of expression and/or synthesis of constitutive adhesion molecules in leukocytes and endothelium, and to interfere in the secretion of pro or anti inflammatory mediators. On the other hand, actions of NO in secreation of chemical mediators by macrophages induces the production of inflammatory mediators and support the duality of NO in the cellular recruitment process.

Inflammatory bowel diseases, (IBD), are a group of chronic disorders of the gastro-intestinal tract, and include Crohn’s disease (CD) and Ulcerative Colitis (UC). The pathogenesis is not known, but involves at least in part a loss of tolerance towards the commensal colonic microbiota. In this thesis, we show in animal models of CD and UC that the colonic mucosal blood flow increased compared to healthy animals. This blood flow increase is due to an up regulation of endothelial nitric oxide synthase (NOS). Further, we show in the UC model that the thickness of the firmly adherent colonic mucus layer increased compared to healthy animals. This increase is due to an up regulation of inducible NOS in the epithelium. Both the blood flow and mucus thickness increase appear to be protective mechanisms.  We demonstrate that the firmly adherent colonic mucus layer acts as a partial barrier towards luminal bacteria. In the UC model, this barrier is destroyed, causing increased bacterial translocation. The adhesion molecule P-selectin was up regulated in the UC model, leading to increased interactions between leukocytes and the endothelium, but also increased interactions between platelets and the endothelium. This indicates that not only leukocytes, but also platelets are involved in colonic inflammation. The addition of the probiotic bacterial strain Lactobacillus reuteri prevented disease by normalizing P-selectin levels and endothelial interactions with leukocytes and platelets. Lactobacillus reuteri also decreased bacterial translocation over the epithelium. In summary, this thesis highlights the importance of colonic barrier functions, and investigates the role of the microbiota in experimental IBD.