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Large vessel vasculitis (LVV), including giant cell arteritis (GCA) and Takayasu arteritis (TAK), is the most common form of primary vasculitis and is characterised by chronic inflammation of medium and large arteries. Accurately determining disease activity in LVV is challenging, particularly once treatment has started. This often leads to a mismatch between disease activity and treatment intensity leaving patients at risk of the consequences of over-treatment, including adverse effects of toxic therapies, or under-treatment, leading to unchecked vascular inflammation and arterial damage. The aim of this thesis was to develop novel methods of assessing disease activity in order to better guide treatment for patients with LVV. The results presented here demonstrate that hybrid positron emission tomography with magnetic resonance imaging (PET/MR) is able to accurately distinguish active from inactive LVV and can track disease activity longitudinally. MR metrics provide complementary information to that provided by PET metrics for determination of disease activity status. A novel, PET/MR-specific disease activity assessment score – the VAMP score – appears more effective at distinguishing active from inactive LVV than established PET-only scoring systems. The imaging data provided by PET/MR have the potential to positively influence ‘real-world’ clinical decision making. Considering that PET/MR utilises a significantly lower radiation dose compared with PET/CT, these results support the use of PET/MR for longitudinal disease monitoring in LVV. This thesis has also demonstrated the efficacy of a series of new and emerging serological biomarkers of disease activity in LVV. Plasma concentrations of leucine-rich alpha-2- glycoprotein-1 (LRG1), angiopoietin-2 (Ang-2), soluble fms-like tyrosine kinase-1 (sFlt-1), osteopontin, and calprotectin are elevated in LVV compared with health. Additionally, LRG1, Ang-2 and osteopontin can distinguish active LVV from inactive LVV, with all biomarkers outperforming C-reactive protein (CRP) for this indication. Correlations were observed between several of the evaluated biomarkers and previously defined measures of disease activity. Finally, I have demonstrated the ability of microcirculatory changes to inform LVV disease activity based on examination of retinal microstructures using optical coherence tomography (OCT). Patients with LVV exhibit chorioretinal thinning which is reversible with treatment. Correlations were also observed between OCT metrics and both PET/MR-based and serological measures of disease activity. These results suggest that microcirculatory dysfunction plays a role in the development and maintenance of inflammation in LVV and may be amenable to therapeutic targeting. Additionally, consideration should be given to the eye as a window through which to define and track disease activity in LVV. In summary, this thesis has identified several ways in which monitoring of disease activity in LVV might be improved. These findings have the potential to enhance LVV patient care by facilitating more accurate matching of treatment intensity with disease activity. Larger, multi- centre studies are now required to confirm and expand on these findings.

Angiopoietin-2 (Ang-2) has been implicated in the development of several types of cancer, including lung malignancy. In the present study, we examined the impact of Ang-2 serum concentration on the development, dissemination, and 5-year overall survival of NSCLC and SCLC. A total of 99 patients with lung cancer were tested. The OS of NSCLC and SCLC patients was estimated using Kaplan–Meier curves and compared through log-rank test. The median serum level of Ang-2 at baseline in both NSCLC and SCLC patients was significantly higher than that of controls (p < 0.0001). The Ang-2 serum concentration was not related to metastasis, neither in NSCLC nor in SCLC cases. The OS was found to be significantly shorter for stage IIIβ NSCLC patients with a high baseline Ang-2 serum concentration (p = 0.012), while Cox regression analysis showed that Ang-2 is a significant independent factor for poor prognosis for stage IIIβ NSCLC (hazard ratio = 2.97, 95% CI: 1.05–8.40, p = 0.04). The concentration of Ang-2 has no impact on the prognosis of SCLC. Ang-2 could be considered as a significant molecular marker that enables the prediction of NSCLC and SCLC development, and is involved in the poor prognosis of stage IIIβ NSCLC.

Abnormal vasculature in tumors leads to poor tissue perfusion and cytostatic drug delivery. Although drugs inducing vascular normalization, for example, angiopoietin-2 (Ang2)-blocking antibodies, have shown promising results in preclinical tumor models, clinical studies have so far shown only little efficacy. Because Ang2 is known to play a protective role in stressed endothelial cells, we tested here whether Ang2 blocking could enhance radiation-induced tumor vascular damage. Tumor-bearing mice were treated with anti-Ang2 antibodies every 3 or 4 days starting 3 days before 3 × 2 Gy or 4 × 0.5 Gy whole-body or tumor-focused radiation. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal tumors. Single-cell RNA-sequencing revealed that Ang2 blocking rescued radiation-induced decreases in T cells and cells of the monocyte/macrophage lineage. In addition, anti-Ang2 enhanced radiation-induced apoptosis in cultured endothelial cells. In vivo, combination treatment decreased tumor vasculature and increased tumor necrosis in comparison with tumors treated with monotherapies. These results suggest that a combination of Ang2-blocking antibodies with radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice. Significance: These findings offer a preclinical rationale for further testing of the use of radiation in combination with Ang2-blocking antibodies to improve the overall outcome of cancer treatment.

Malaria-related deaths could be prevented if powerful diagnostic and reliable prognostic biomarkers were available to allow rapid prediction of the clinical severity allowing adequate treatment. Using quantitative ELISA, we assessed the plasma concentrations of Procalcitonin, Pentraxine-3, Ang-2, sTie-2, suPAR, sEPCR and sICAM-1 in a cohort of Beninese children with malaria to investigate their potential association with clinical manifestations of malaria. We found that all molecules showed higher levels in children with severe or cerebral malaria compared to those with uncomplicated malaria (p-value < 0.005). Plasma concentrations of Pentraxine-3, Procalcitonin, Ang-2 and the soluble receptors were significantly higher in children with coma as defined by a Blantyre Coma Score < 3 (p < 0.001 for Pentraxine-3, suPAR, and sTie-2, p = 0.004 for PCT, p = 0.005 for sICAM-1, p = 0.04 for Ang-2). Moreover, except for the PCT level, the concentrations of Pentraxine-3, suPAR, sEPCR, sICAM-1, sTie-2 and Ang-2 were higher among children who died from severe malaria compared to those who survived (p = 0.037, p = 0.035, p < 0.0001, p= 0.0008, p = 0.01 and p = 0.02, respectively). These findings indicate the ability of these molecules to accurately discriminate among clinical manifestations of malaria, thus, they might be potentially useful for the early prognostic of severe and fatal malaria, and to improve management of severe cases.

Moyamoya arteriopathy (MMA) is a rare cerebrovascular disorder that causes recurrent ischemic and hemorrhagic strokes, leading young patients to severe neurological deficits. The pathogenesis of MMA is still unknown. The disease onset in a wide number of pediatric cases raises the question of the role of genetic factors in the disease’s pathogenesis. In these patients, MMA’s clinical course, or progression, is largely unclear. By performing a comprehensive molecular and cellular profile in the plasma and CSF, respectively, of MMA pediatric patients, our study is aimed at assessing the levels of circulating endothelial progenitor cells (cEPC) and the release of selected proteins at an early disease stage to clarify MMA pathogenesis and progression. We employed cytofluorimetric methods and immunoassays in pediatric MMA patients and matched control subjects by age and sex. We detected increased levels of cEPC in peripheral blood and an upregulation of angiogenic markers in CSF (i.e., angiopoietin-2 and VEGF-A). This finding is probably associated with deregulated angiogenesis, as stated by the moderate severity of collateral vessel network development (Suzuki III-IV). The absence of significant modulation of neurofilament light in CSF led us to rule out the presence of substantial neuronal injury in MMA children. Despite the limited cohort of pediatric patients, we found some peculiar cellular and molecular characteristics in their blood and CSF samples. Our findings may be confirmed by wider and perspective studies to identify predictive or prognostic circulating biomarkers and potential therapeutic targets for personalized care of MMA pediatric patients.

Non-alcoholic fatty liver disease is the most common liver disorder worldwide, and its progressive form non-alcoholic steatohepatitis (NASH) is a growing cause of liver cirrhosis and hepatocellular carcinoma (HCC). Lifestyle changes, which are capable of improving the prognosis, are hard to achieve, whereas a pharmacologic therapy able to combine efficacy and safety is still lacking. Looking at the pathophysiology of various liver diseases, such as NASH, fibrosis, cirrhosis, and HCC, the process of angiogenesis is a key mechanism influencing the disease progression. The relationship between the worsening of chronic liver disease and angiogenesis may suggest a possible use of drugs with antiangiogenic activity as a tool to stop or slow the progression of the disorder. In this review, we highlight the available preclinical data supporting a role of known antiangiogenic drugs (e.g., sorafenib), or phytotherapeutic compounds with multiple mechanism of actions, including also antiangiogenic activities (e.g., berberine), in the treatment of NASH.

Xiaofeng Xu,1,* Yuhua Yan,1,2,* Qingying Xun,3 Jiayu Shi,1,4 Xiangyi Kong,1 Jun Wu,1 Huaijun Zhou1 1Department of Gynecology, Nanjing Drum Tower Hospital, Nanjing University Medical School, Nanjing, Jiangsu 210008, China; 2Department of Gynecology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi 530021, China; 3Department of Physiology, Medical College, Southeast University, Nanjing, Jiangsu 210009, China; 4Department of Obstetrics and Gynecology, The First Affiliated Hospital of Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450008, China *These authors contributed equally to this work Background: Angiogenesis is critical for the growth and metastasis of solid tumors and is, therefore, an important therapeutic target. Despite the great research advances in tumor therapies targeting vascular endothelial growth factor (VEGF), drug resistance frequently occurs, and further strategies targeting the tumor vasculature are of primary concern. Purpose: The present study aimed to determine whether a combination of small interfering RNAs (siRNAs) targeting VEGF-A and angiopoietin-2 (Ang-2) inhibited the biologic mechanisms of endometrial cancer more effectively compared to either one alone, in vitro and in vivo. Methods: VEGF-A and Ang-2 were silenced by siRNA in Ishikawa endometrial cancer cells. Cell growth, apoptosis, metastasis, and tumor angiogenesis were measured in vitro and in vivo. Results: There was no difference observed in cell apoptosis rate; however, combined silencing of VEGF-A and Ang-2 resulted in a stronger inhibition of cell proliferation and invasion (P

Highlights • The ECM Multimerin-2 is a substrate for pericyte adhesion. • The recruitment of pericytes leads to enhanced Multimerin-2 expression by endothelial cells. • Multimerin-2 induces the expression of important cytokines both in endothelial cells and pericytes. • The deposition of Multimerin-2 is key for the endothelial cell/pericyte crosstalk required for the establishment of vascular stability., Tumor angiogenesis is vital for the growth and development of various solid cancers and as such is a valid and promising therapeutic target. Unfortunately, the use of the currently available anti-angiogenic drugs increases the progression-free survival by only a few months. Conversely, targeting angiogenesis to prompt both vessel reduction and normalization, has been recently viewed as a promising approach to improve therapeutic efficacy. As a double-edged sword, this line of attack may on one side halt tumor growth as a consequence of the reduction of nutrients and oxygen supplied to the tumor cells, and on the other side improve drug delivery and, hence, efficacy. Thus, it is of upmost importance to better characterize the mechanisms regulating vascular stability. In this context, recruitment of pericytes along the blood vessels is crucial to their maturation and stabilization. As the extracellular matrix molecule Multimerin-2 is secreted by endothelial cells and deposited also in juxtaposition between endothelial cells and pericytes, we explored Multimerin-2 role in the cross-talk between the two cell types. We discovered that Multimerin-2 is an adhesion substrate for pericytes. Interestingly, and consistent with the notion that Multimerin-2 is a homeostatic molecule deposited in the later stages of vessel formation, we found that the interaction between endothelial cells and pericytes promoted the expression of Multimerin-2. Furthermore, we found that Multimerin-2 modulated the expression of key cytokines both in endothelial cells and pericytes. Collectively, our findings posit Multimerin-2 as a key molecule in the cross-talk between endothelial cells and pericytes and suggest that the expression of this glycoprotein is required to maintain vascular stability.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Background Forkhead box C2 (FOXC2) is a crucial factor involving in various cancers. However, its functions in hepatocellular carcinoma (HCC) is unknown. Here, we explored the role of FOXC2 in the progression of HCC and its potential mechanisms. Methods FOXC2 expression in HCC tissue and cells were detected by immunohistochemistry or western blot and real-time PCR. CCK8, wound healing and transwell assay were used to measure cell growth and invasion. Tumor formation experiment was carried out to assess the tumorigenicity of HCC cells. Regulation of FOXC2 on Ang-2 was validated by luciferase assay and complementary experiments. Results Increased FOXC2 expression was found to be associated positively with more aggressive clinicopathologic features. HCC patients with higher FOXC2 expression had significantly shorter overall survival. FOXC2 expression was indentified as an independent risk factor for resectable HCC. Increased FOXC2 expression accelerated the migration and invasion of HCC cells, accompanied by enhanced Ang-2 expression. Likewise, FOXC2 knockdown yielded opposite results. Moreover, FOXC2 stimulated the activation of the Ang-2 promoter. Suppression of Ang-2 expression hindered the FOXC2-mediated EMT processs, cell migration and invasion of HCC. Conclusions FOXC2 is a novel prognostic predictor for HCC and may facilitate the growth and invasion through Ang-2.

IgA nephropathy (IgAN) is recognized as most frequent form of primary glomerulonephritis worldwide. IgAN is associated with renal degradation occurring due to irreversible pathological changes leading to glomerulosclerosis and interstitial fibrosis. It remains poorly understood whether and to what extent these changes are followed by the activation of regenerative mechanisms. Therefore, in this study we aimed to evaluate regenerative potential of IgAN patients by quantitating the frequencies of several stem cell types, namely circulating very small embryonic-like stem cells (VSELs), hematopoietic stem cells (HSCs), endothelial progenitor cells (EPCs) as well as different monocyte subsets with varying maturation and angiopoietic potential. Moreover, we analyzed whether changes in stem cell and monocyte frequencies were related to alterations of several chemotactic factors (stromal derived-factor (SDF-1), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2)) and a marker of monocyte/macrophage activation, namely soluble form of CD163 receptor (sCD163). We showed that IgAN patients presented with enhanced levels of VSELs, but not other stem cell types. We also demonstrated significantly elevated numbers of intermediate monocytes known for their M2-like properties as well as high angiopoietic potential and CD163 expression. This finding was accompanied by detection of elevated sCD163 plasma levels in IgAN patients. Taking together, we demonstrated here that IgAN is associated with selective mobilization of VSELs and increased maturation of monocytes towards M2-like and angiopoietic phenotype. These findings contribute to better understanding of the role of regenerative mechanisms in the pathogenesis of chronic inflammation in the course of IgAN.

Angiogenesis is a biological process that involves the formation of new blood vessels from the existing vasculature, and it plays a fundamental role in the development and progression of several types of cancer, including lung cancer. The angiopoietin/Tie2 ligand/receptor system orchestrates vascular integrity. In particular, Angiopoietin-1 activates the endothelial cell (EC)-specific receptor tyrosine kinase,Tie2,which is essential for preserving endothelial quiescence. On the other hand, Angiopoietin-2 acts as an inhibitor of the Angiopoietin-1/Tie2 signaling pathways, thus facilitating the destabilization of quiescent endothelium in cases of inflammation and cancer. Clinical studies have proven that high levels of Angiopoietin-2 indicate the development of non-small-cell lung carcinomas (NSCLC), while high levels of Angiopoietin-2 are strongly related to tumor angiogenesis, lymphangiogenesis, metastasis, and poor prognosis. Interestingly, the association of Angiopoietin-2 levels with the type of surgical approach makes Angiopoietin-2 a valuable factor in selecting the most suitable therapeutic strategy for lung cancer patients. The role of the Angiopoietin-1 and Angiopoietin-4 levels in NSCLC development requires further investigation. The present review focuses on the clinical impact of the Angiopoietin-1, Angiopoietin-2, and Angiopoietin-4 levels in patients diagnosed with NSCLC, emphasizing the interaction between them, and how they affect the development, progression, and metastasis of lung disease. Finally, it estimates the role of angiopoietins levels in the effective therapy of lung cancer patients.

Lung cancer is the most common malignant tumor with increasing angiopoietin-2 (Ang-2) and a high rate of metastasis. However, the mechanism of Ang-2 enhancing tumor proliferation and facilitating metastasis remains to be clarified. In this study, Ang-2 expression and its gene transcription on effects of biological behaviors and epithelial-mesenchymal transition (EMT) were investigated in lung cancers. Total incidence of Ang-2 expression in the cancerous tissues was up to 91.8 % (112 of 122) with significantly higher (χ2=103.753, P2=7.883, P=0.005), differentiation degree (χ2=4.554, P=0.033), tumor node metastasis (TNM) staging (χ2=5.039, P=0.025), and 5-year survival rate (χ2 =11.220, P2=18.881, P2=0.81, P=0.776) or III & IV (χ2=1.845, P=0.174). Over-expression of Ang-2 or Ang-2 mRNA in lung A549 and NCI-H1975 cells were identified among different cell lines. When silencing Ang-2 in A549 cells with specific shRNA-1 transfection, the cell proliferation was significantly inhibited in a time-dependent manner, with up-regulating E-cadherin, down-regulating Vimentin, Twist, and Snail expression, and decreasing invasion and metastasis of cancer cell abilities, suggesting that Ang-2 promote tumor metastasis through increasing EMT, and it could be a potential target for lung cancer therapy.

PURPOSE: Vascular inflammation and leakage in sepsis is mediated by Angiopoietin-1 (Ang-1) and -2 (Ang-2) and their phosphorylation of the endothelial Tie-2 receptor. Levels of Ang-2 change in adults and children during sepsis, which is associated with severity of sepsis. This study investigates levels of Ang-1 and Ang-2 in newborns to gain insight in the vascular pathophysiology of early onset sepsis (EOS) within 72 hours after birth.METHODS: A prospective cohort study was performed amongst 71 Surinamese newborns treated with antibiotics for suspected EOS and 20 control newborns. Newborns with suspected EOS were divided in two groups: blood culture negative and positive EOS. Ang-1 and Ang-2 levels were measured in serum obtained at start of antibiotic treatment and at reevaluation after 48-72 hours.RESULTS: In this cohort 8.5% of newborns had a positive blood culture. At start of antibiotic treatment Ang-1 serum levels were lower (P CONCLUSIONS: Our findings support the hypothesis that a dysbalance in the Angiopoietins plays a role in the vascular pathophysiology of EOS.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. http://creativecommons.org/licenses/by-nc-nd/4.0.

Vascular endothelial growth factor (VEGF) inhibition by the addition of bevacizumab to the chemotherapy regimen of metastatic colorectal cancer leads to an improved outcome. However, anti-angiogenic tumor therapy targeting a single factor may be limited by complementary mechanisms. Angiopoietin-2 (Ang-2, ANGPT2) is another important factor that cooperates with VEGF to drive tumor angiogenesis. It was shown that high Ang-2 levels are associated with a poor clinical outcome of colorectal cancer patients treated with bevacizumab-containing therapy. Therefore, combined inhibition of VEGF and Ang-2 was supposed to improve anti-angiogenic therapy. Here, we evaluated the efficacy of a bispecific antibody (CrossMab) co-targeting VEGF and Ang-2 in combination with chemotherapy in a chemoresistant colorectal carcinoma model. Antitumor activity was evaluated in athymic nude mice bearing subcutaneous DLD1 xenograft tumors and treated with anti-VEGF (B20), anti-Ang-2 (LC06) and anti-VEGF/Ang-2 (CrossMab) antibodies. Chemotherapy consisted of 5-FU and irinotecan. Resected tumors were analyzed immunohistochemically. First, an impact of targeting each single factor but also a clear advantage of co-targeting both factors could be demonstrated. Accordingly, tumor tissue showed strong staining for VEGF and Ang-2. Chemotherapy alone was less effective. Efficient tumor growth inhibition could be achieved by treatment with anti-VEGF/chemotherapy, single CrossMab and CrossMab/chemotherapy, which resulted in 3 out of 10, 6 out of 10 and 10 out of 10 complete responses, respectively, during seven weeks. Complete retarded tumors were characterized by massive intratumoral necrosis surrounded by layers of vital tumor cells and connective tissue with CD31-positive vessels at the periphery. In some cases, a distinct feature known as vessel co-option could be observed. In conclusion, the data from this model clearly support the strategy of co-targeting VEGF and Ang-2 and further demonstrate the beneficial impact of co-treatment with chemotherapy. The clear superiority of the CrossMab-containing regimen compared to clinical standard anti-VEGF/chemotherapy warrants further analyses in other models.

Purpose Corneal transplantation remains the last hope for vision restoration, and lymphangiogenesis (LG) is a primary mediator of transplant rejection. This study was to investigate the specific role of angiopoietin-2 (Ang-2) in transplantation-associated LG and graft rejection. Methods Orthotopic corneal transplantation was performed between fully mismatched C57BL/6 (donor) and BALB/c (recipient) mice to assess the effects of Ang-2 blockade via neutralizing antibody. Grafts were evaluated in vivo by ophthalmic slit-lamp biomicroscopy and anterior segment optical coherence tomography (OCT) up to 8 weeks after surgery. Additionally, whole-mount corneas were analyzed for lymphatic and blood vessels and macrophages by immunofluorescent microscopy, and draining lymph nodes were assessed for donor-derived cells by flow cytometry. Results Anti-Ang-2 treatment significantly suppressed LG and graft rejection. In this study, we achieved 75% suppression of LG and 80% graft survival. Our approach also inhibited donor-derived cell trafficking to draining lymph nodes and affected macrophage morphologic phenotypes in the grafted corneas. Additionally, Ang-2 blockade also reduced central corneal thickening, a parameter strongly associated with graft rejection. Conclusions Ang-2 is critically involved in corneal transplant rejection and anti-Ang-2 treatment significantly improves the outcomes of corneal grafts. Moreover, we have shown that anterior segment OCT offers a new tool to monitor murine corneal grafts in vivo. This study not only reveals new mechanisms for transplant rejection, but also offers a novel strategy to treat it.

Sepsis represents one the main cause of death in patients admitted to the intensive care. Biomarkers offer an alternative approach to the diagnostic and prognostic evaluation and improve the outcomes. Angiopoietin 2 (Ang-2) and Tyrosine kinase 2 (Tie-2) are biomarkers which may be involved in sepsis, Ang-2 being responsible for vascular remodelling while Tie-2 is their endothelial receptor. The aim of the study: To assess the Ang-2, Tie-2 and Ang-2/Tie-2 ratio serum levels in septic and non-septic patients and to investigate the independent value of circulating Ang-2, Tie-2, and Ang-2/Tie-2 ratios as predictors of prognosis in critically ill medical patients. Study design: The study included 74 adults admitted to an intensive care unit (ICU). The patients were separated in two groups: Group A [sepsis: n=40] and Group B [no-sepsis: n= 34] patients. Serum levels of Ang-2 and Tie-2 were determined in the first 12 hours after admission and were correlated with ICU severity scores, APACHE II, SOFA and SAPS and with the death rate. Results: Group A gave significantly higher values (p=0.01), for serum Ang-2 (11.07±9.21 ng/ml) compared to Group B (6.18±5.28 ng/ml). The level of Tie-2 was also higher (11.03±5.12 ng/ml) in Group A compared to Group B (9.46±4.99 ng/ml) (p=0.19). In Group A, the Ang-2/Tie2 ratio showed higher values than Group B (p=0.02). There was a positive association between severity scores (APACHE II, SAPS, and SOFA) and Ang-2, and Ang-2/ Tie-2 ratio, but not for Tie2. Conclusions: In our study Ang-2 and Ang-2/Tie-2 ratio serum levels had independent diagnostic value in patients with sepsis, as measured on admission.

Age-related macular degeneration (AMD) constitutes a prevalent, chronic, and progressive retinal degenerative disease of the macula that affects elderly people and cause central vision impairment. Despite therapeutic advances in the management of neovascular AMD, none of the currently used treatments cures the disease or reverses its course. Medical treatment of neovascular AMD experienced a significant advance due to the introduction of vascular endothelial growth factor inhibitors (anti-VEGF), which dramatically changed the prognosis of the disease. However, although anti-VEGF therapy has become the standard treatment for neovascular AMD, many patients do not respond adequately to this therapy or experience a slow loss of efficacy of anti-VEGF agents after repeated administration. Additionally, current treatment with intravitreal anti-VEGF agents is associated with a significant treatment burden for patients, caregivers, and physicians. New approaches have been proposed for treating neovascular AMD. Among them, designed ankyrin repeat proteins (DARPins) seem to be as effective as monthly ranibizumab, but with greater durability, which may enhance patient compliance with needed injections.

Made available in DSpace on 2019-10-06T16:49:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Introduction: Preeclampsia affects 3–5% of pregnancies worldwide and is the primary cause of maternal-fetal and neonatal mortality. Previous studies show that alterations in maternal concentrations of angiogenic factors, such as PlGF, PDGF AA, ANG-1, and ANG-2, may play fundamental roles in the pathophysiology of the disease. Objective: Determine whether the PlGF, PDGF AA, ANG-1, and ANG-2 are predictors of preeclampsia occurrence in a prenatal cohort study. Patients and methods: This is a case-control study associated with a prospective cohort of pregnant women, with gestational ages between 20 and 25 weeks, composed of 30 pregnant women with preeclampsia (PE) and 90 healthy pregnant women (HP). The plasma concentrations of the markers were determined using the ELISA method. The comparison between the case and control groups was performed using the t test on the SAS® 9.4 software. Also, ROC curves were constructed to evaluate the predictive potential of the biomarkers. Results: Differences in the concentrations of PlGF, PDGF AA, ANG-1 and ANG-2, and the ANG-1/ANG-2 ratio were not observed between the PE and the HP groups. The predictive capacity of the biomarkers was assessed using ROC curves, in which the area under the curve for PlGF AUC = 0.55; PDGF AA AUC = 0.55; ANG-1 AUC = 0.47; ANG-2 AUC = 0.51, and the ANG-1/ANG-2 ratio AUC = 0.57. Conclusion: In pregnant women, with gestational ages between 20 and 25 weeks significant differences in biomarker concentrations between groups PE and HP were not observed. The ROC curves showed that the biomarkers were ineffective as preeclampsia predictors in the analyzed cohort. Department of Gynecology and Obstetrics Ribeirão Preto Medical School University of São Paulo, 3900 Bandeirantes Avenue Department of Health Sciences Ribeirão Preto Medical School University of São Paulo, 3900 Bandeirantes Avenue Department of Pharmacology Biosciences Institute State University of São Paulo (UNESP) Department of Childcare and Pediatrics Ribeirão Preto Medical School University of São Paulo, 3900 Bandeirantes Avenue Department of Pharmacology Biosciences Institute State University of São Paulo (UNESP) FAPESP: FAPESP

Objective Determine the effect of bradykinin on solute permeability and cellular junctional proteins in human dermis microvascular endothelial cells. Methods Cells were characterized by immunofluorescence and fluorescence���activated cell sorting. Macromolecular transport of dextran and albumin was monitored. Junctional protein expression and phosphorylation were determined by immunoblot analyses. Intracellular calcium and cAMP levels were evaluated. Target gene expression at mRNA and protein levels was determined. Results Human dermis microvascular endothelial cells comprised 97% lymphatic endothelial cells. Bradykinin increased the permeability to dextran in a concentration���dependent manner, while reduced the permeability to albumin. Bradykinin treatment down���regulated VE���cadherin expression and affected its phosphorylation status at Tyr731. It also down���regulated claudin���5 expression at the transcriptional level through bradykinin���2���receptor signaling. An increase in the intracellular calcium levels and a reduction in the cAMP concentration were associated effects. Finally, bradykinin induced the up���regulation of vascular endothelial growth factor���C protein which was found increased in BK���induced human dermis microvascular endothelial cells culture supernates. Conclusions Human dermis microvascular endothelial cells represent a model of lymphatic endothelial cells, in which bradykinin���2���receptor is expressed. Bradykinin���induced bradykinin���2���receptor signaling through intracellular calcium mobilization and reduction in cAMP levels, triggered changes in solute permeability and cellular junction expression. It further up���regulated vascular endothelial growth factors���C protein expression, which is a key modulator of lymphatic vessels function and lymphangiogenesis, publishedVersion

The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

Background Lung cancer is the most incident and lethal form of cancer, with late diagnosis as a major determinant of its bad prognosis. Immunotherapies targeting immune checkpoints improve survival, but positive results encompass only 30%-40% of the patients, possibly due to alternative pathways to immunosuppression, including tumour-associated macrophages (TAM). Colony stimulating factor-1 (CSF-1) is implicated in TAM differentiation and recruitment to tumours and in tumour angiogenesis, through a special setting of Tie-2-expressing macrophages, which respond to angiopoietin-2 (Ang-2). We evaluated the role of serum levels of CSF-1 in non-small cell lung cancer (NSCLC) prognosis and whether these could serve as biomarkers for NSCLC detection, along with Ang-2. Participants and methods We prospectively studied an unselected cohort of 145 patients with NSCLC and a group of 30 control individuals. Serum levels of Ang-2 and CSF-1 were measured by ELISA prior to treatment. Results Serum levels of CSF-1 and Ang-2 are positively correlated (p

Objective: Carcinoma of the uterine cervix is either the first or second most common malignancy in Indian women, depending on the registry. Tumor growth and metastasis primarily are determined by angiogenesis and parameters of the molecular environment including extracellular matrix elements, growth factors and cytokines. Effects of chemo-irradiation on biomarkers like vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2) and laminin in patients with carcinoma cervix therefore need to be explored. Methods: Circulatory and mRNA levels of VEGF, Ang-2 and laminin in patients with stage III carcinoma cervix (n=40) were compared with those of normal healthy women (n=20). Measurement was prior to treatment, and after chemotherapy and teleradiation, using high sensitivity ELISA kits and Q-PCR. Clinical response was evaluated as per WHO criteria and was assessed for correlation with the biochemical markers. Results: Levels of all the studied molecules were significantly (p

Objective: To detect the expression of Ang-2 and VEGF in the endometriosis (EMT), and to explore the correlation of Ang-2 and VEGF with the pathogenesis of EMT. Methods: A total of 60 patients with EMT who were confirmed by laparoscopy and admitted in our hospital from August, 2014 to August, 2015 were included in the study and served as the observation group; moreover, 60 women who came for laparoscopy at the same stage were served as the control group. A volume of 5mL morning fasting elbow venous blood in the two groups was extracted, standing in the room temperature for 1h, centrifuged at 3 500 r/min for 20 min. The supernatant was extracted and stored at -70 ℃ for inspection. During the laparoscopy, a volume of 5 mL peritoneal fluid was collected, standing in the room temperature for 1 h, centrifuged at 3 500 r/min for 20 min. The supernatant was extracted and stored at -70 ℃ for inspection. ELISA was used to detect the levels of Ang-2 and VEGF. Results: The levels of Ang-2 and VEGF in the serum and peritoneal fluid in the observation group were significantly higher than those in the control group (P

Objective The aim of this study was to investigate the effects of docosahexaenoic acid (DHA) on the generation of angiopoietin-2 (Ang-2) by rat brain microvascular endothelial cells under an oxygen- and glucose-deprivation environment (OGD), and its relationship, if any, with cyclooxygenase 2 (COX-2) expression. Methods Annexin V and propidium iodide apoptosis assay was used to detect apoptosis. Enzyme linked immunosorbent assay was used to detect Ang-2, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and prostaglandin I2 (PGI2) content. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect Ang-2 and VEGF mRNA expression. Western blot was used to detect expression of COX-2 protein. Results DHA reduced the apoptosis rate (P = 0.026) and decreased the secretion of Ang-2, VEGF, PGE2, and PGI2 (P = 0.006, P = 0.000, P = 0.002, P = 0.004 respectively). The relative expression of Ang2 and Vegf mRNA, as well as COX-2 expression, also decreased (P = 0.000, P = 0.005, P = 0.007 respectively). These effects were antagonized by GW9662 (peroxisome proliferator-activated receptor-γ antagonist). COX-2 protein expression levels were positively correlated with Ang2 and Vegf mRNA expression levels (γ = 0.69, P = 0.038 and γ = 0.76, P = 0.032, respectively). Ang-2 and VEGF mRNA levels were positively correlated with Ang-2 (γ = 0.84, P = 0.012) and VEGF (γ = 0.71, P = 0.036) secretion levels respectively. Conclusion DHA reduced apoptosis induced by an OGD environment, thus decreasing Ang-2 and VEGF synthesis. This phenomenon was associated with a decrease in COX-2 protein expression, PGE2 and PGI2 secretion, and generation regulation via intracellular transcriptional pathways.

Background Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide. The targeted therapy had made important progress in recent years, but few potential predictive biomarkers for prognosis of NSCLC patients were identified. Angiopoietin-2 (Ang-2), a cytokine upregulated in tumor endothelial cells and some tumor cells including NSCLC, is a partial agonist and antagonist of angiopoietin-1 (Ang-1). Ang-1 is another ligand for the tyrosine kinase receptor Tie2; it promotes recruitment of pericytes and smooth muscle cells, stabilizing vascular networks by binding to Tie2. Although many studies mainly considered that Ang-2 correlated with progression and prognosis of NSCLC significantly, there are much conflicting and controversial data. Therefore, we conducted a meta-analysis to assess the relationship between Ang-2 and prognosis, a clinical outcome of NSCLC. Methods The search was based on major databases from PubMed, Cochrane Library, EMBASE, and CNKI, and 20 eligible publications (range from 2002 to 2015) are included in our meta-analysis with 2011 NSCLC patients in total. These studies illuminated the correlation between the expression of Ang-2 and NSCLC, based on either prognostic factors or clinicopathological features. Pooled calculations were carried out on the odds ratio (OR) and the corresponding 95 % confidence interval (CI) to perform this meta-analysis, and all statistical analyses were carried out by STATA 12.0 and Review Manager 5.3. Results According to our results, the expression of Ang-2 in NSCLC tissues was significantly higher than that in normal lung tissues, indicating that Ang-2 over-expression may be a predictive marker (pooled OR = 5.09, corresponding 95 % confidence interval (95 % CI) 3.10–8.36, p = 0.000). In addition, our pooled data showed that Ang-2 expression was positively correlated with tumor stages (pooled OR = 3.58, 95 % CI 2.40–5.35, p = 0.000), differentiation (pooled OR = 0.65, 95 % CI 0.45–0.94, p = 0.02), lymphatic invasion (pooled OR = 3.15, 95 % CI 1.97–5.03, p = 0.000), and poor survival (pooled OR = 1.93, 95 % CI 1.47–2.52, p = 0.000) of NSCLC, but seems to have no significant impact on tumor size (pooled OR = 1.09, 95 % CI 0.59–2.00, p = 0.78). Conclusions These results demonstrate that Ang-2 expression significantly correlated with poor prognosis for patients with NSCLC.

Background: To assess antivascular effects, and evaluate clinically translatable magnetic resonance imaging (MRI) biomarkers of tumour response in vivo, following treatment with vanucizumab, a bispecific human antibody against angiopoietin-2 (Ang-2) and vascular endothelial growth factor-A (VEGF-A). Methods: Colo205 colon cancer xenografts were imaged before and 5 days after treatment with a single 10 mg kg−1 dose of either vanucizumab, bevacizumab (anti-human VEGF-A), LC06 (anti-murine/human Ang-2) or omalizumab (anti-human IgE control). Volumetric response was assessed using T2-weighted MRI, and diffusion-weighted, dynamic contrast-enhanced (DCE) and susceptibility contrast MRI used to quantify tumour water diffusivity (apparent diffusion coefficient (ADC), × 106 mm2 s−1), vascular perfusion/permeability (Ktrans, min−1) and fractional blood volume (fBV, %) respectively. Pathological correlates were sought, and preliminary gene expression profiling performed. Results: Treatment with vanucizumab, bevacizumab or LC06 induced a significant (P

Background: Gastro-entero-pancreatic/neuroendocrine (NET) tumors are highly vascularized neoplasms. However, our knowledge concerning circulating levels of the angiogenic factors in NET patients still remains insufficient. Methods: The aim of this study was to measure plasma concentrations of VEGF, angiopoietin 1 (Ang-1), angiopoietin 2 (Ang-2), soluble Tie-2, endostatin, osteopontin (OPN) and chromogranin A (CgA) in 36 NET patients and 16 controls. Results: Only the plasma concentrations of Tie-2 and CgA were higher in NET patients as compared to controls. These levels were within the reference range in controls; however one control demonstrated slightly elevated Tie-2 and 4 elevated CgA. Similarly, in the subgroup of patients with carcinoid syndrome, only Tie-2 and CgA concentrations were higher than those in patients with non-functioning NETs. In turn, in the subgroup of metastatic patients, only Ang-2 levels were higher than in those with localized disease. A positive correlation was found between Ang-2 and Tie-2 levels in metastatic patients and between Ang-1 and Tie-2 in localized NETs. Conclusions: The plasma concentration of Tie-2 is proposed as an additional marker for NET patients and seems to be similarly effective as the currently used CgA level. Moreover, higher plasma levels of Ang-2 together with the positive correlation between Ang-2 and Tie-2 levels in metastatic subjects, implies that cases with a Tie-2 level above the upper limits, together with higher level of Ang-2 seem to be highly predictive of metastases.

Backgrounds/Aims: Pericyte loss, vasoregression and neuroglial activation are characteristic changes in incipient diabetic retinopathy. In this study, the effect of the antioxidant and antiglycating dipeptide carnosine was studied on the development of experimental diabetic retinopathy. Materials/Methods: STZ-induced diabetic Wistar rats were orally treated with carnosine (1g/kg body weight/day). Retinal vascular damage was assessed by quantitative morphometry. Retinal protein extracts were analyzed for markers of oxidative stress, AGE-formation, activation of the hexosamine pathway and changes in the expression of Ang-2, VEGF and heat shock proteins Hsp27 and HO-1. Glial cell activation was analyzed using Western blot analysis and immunofluorescence of GFAP expression and retinal neuronal damage was histologically examined. Results: Oral carnosine treatment prevented retinal vascular damage after 6 months of experimental hyperglycemia. The protection was not caused by ROS-or AGE-inhibition, but associated with a significant induction of Hsp27 in activated glial cells and normalization of increased Ang-2 levels in diabetic retinas. A significant reduction of photoreceptors in retinas of carnosine treated animals was noted. Conclusion: Oral carnosine treatment protects retinal capillary cells in experimental diabetic retinopathy, independent of its biochemical function. The vasoprotective effect of carnosine might be mediated by the induction of protective Hsp27 in activated glial cells and normalization of hyperglycemia-induced Ang-2. Copyright (C) 2011 S. Karger AG, Basel

Angiogenesis plays a critical role in the development and growth of solid tumors and hematologic malignancies. The system involving angiopoietin-2 [Ang-2] and its receptor Tie-2 appears to play an important role in tumor angiogenesis and in the biology of hematological and non-hematological malignancies. We evaluated the levels of soluble (s)Ang-2 and sTie-2 in acute myeloid leukemia (AML) patients and investigated the impact of their circulating levels on the overall survival in those patients.Ang-2 and Tie-2 were measured in plasma samples from AML patients and controls using enzyme-linked immunosorbent assay (ELISA).The levels of sAng-2 and sTie-2 were significantly higher in AML patients (2382.1±1586.1 pg/ml and 6.74±3.47 ng/ml, respectively) than in controls (649.5±402.6 pg/ml and 2.63±0.57 ng/ml, respectively; p0.01). AML patients with high levels of sAng-2 and sTie-2 (≥2500 pg/ml and ≥8 ng/ml, respectively) had significantly shorter overall survival than those patients with low levels (2500 pg/ml and8 ng/ml, respectively).The results of our study demonstrated the prognostic significance of circulating sAng-2 and sTie-2 in AML patients. Modulation of the angiopoietin / Tie-2 axis may be a promising approach to improve the outcome in those patients.AMAÇ: Anjiyojenez, katı tümörler ve hematolojik hastalıkların gelişimi ve büyümesinde kritik bir rol oynamaktadır. Anjiyopoietin-2 [Ang-2] ve onun reseptörü Tie-2’yi kapsayan sistemin, tümör anjiyojenezinde ve hematolojik ve hematoloji dışı hastalıkların biyolojisinde önemli bir rol oynadığı görülmektedir. AML hastalarında çözünebilen sAng-2 ve sTie-2 düzeyleri değerlendirilmiş olup, bu hastalarda dolaşımdaki düzeylerinin toplam sağkalım üzerindeki etkileri araştırılmıştır. YÖNTEMLER: AMLli hastalar ve kontrollerden alınan plazma örneklerinde, Enzim bağlı immünassay (ELISA) ile Ang-2 ve Tie -2 ölçülmüştür.AML hastalarında, sAng-2 ve sTie-2 değerleri [sırasıyla 2382.1±1586.1 pg/ml ve 6.74±3.47ng/ml], kontrollere [sırasıyla 649.5±402.6 pg/ml ve 2.63±0.57 ng/ml] kıyasla anlamlı derecede daha yüksekti (p0.01). Toplam sağkalım değeri,yüksek sAng-2 ve sTie-2 [sırasıyla ≥2500pg/ml ve ≥8ng/ml] değerli AML hastalarında düşük düzeylere [SONUÇ: Çalışmada elde edilen sonuçlarda, AML hastalarının dolaşımdaki Ang-2 ve sTie-2 düzeylerinin prognostik anlamlılığı gösterilmiştir. Anjiyopoietin / Tie2 aksisinin modülasyonu, söz konusu hastalarda sonucu geliştirmeye yönelik ümit verici bir yaklaşım olabilir.

Objective Lung injury activates multiple pro-inflammatory pathways, including neutrophils, epithelial, and endothelial injury, and coagulation factors leading to acute respiratory distress syndrome (ARDS). Low-dose methylprednisolone therapy (MPT) improved oxygenation and ventilation in early pediatric ARDS without altering duration of mechanical ventilation or mortality. We evaluated the effects of MPT on biomarkers of endothelial [Ang-2 and soluble intercellular adhesion molecule-1 (sICAM-1)] or epithelial [soluble receptor for activated glycation end products (sRAGE)] injury, neutrophil activation [matrix metalloproteinase-8 (MMP-8)], and coagulation (plasminogen activator inhibitor-1). Design Double-blind, placebo-controlled randomized trial. Setting Tertiary-care pediatric intensive care unit (ICU). Patients Mechanically ventilated children (0–18 years) with early ARDS. Interventions Blood samples were collected on days 0 (before MPT), 7, and 14 during low-dose MPT (n = 17) vs. placebo (n = 18) therapy. The MPT group received a 2-mg/kg loading dose followed by 1 mg/kg/day continuous infusions from days 1 to 7, tapered off over 7 days; placebo group received equivalent amounts of 0.9% saline. We analyzed plasma samples using a multiplex assay for five biomarkers of ARDS. Multiple regression models were constructed to predict associations between changes in biomarkers and the clinical outcomes reported earlier, including P/F ratio on days 8 and 9, plateau pressure on days 1 and 2, PaCO2 on days 2 and 3, racemic epinephrine following extubation, and supplemental oxygen at ICU discharge. Results No differences occurred in biomarker concentrations between the groups on day 0. On day 7, reduction in MMP-8 levels (p = 0.0016) occurred in the MPT group, whereas increases in sICAM-1 levels (p = 0.0005) occurred in the placebo group (no increases in sICAM-1 in the MPT group). sRAGE levels decreased in both MPT and placebo groups (p

Hintergrund: Die PH ist eine schwerwiegende und fortschreitende Erkrankung, für die es bislang keine kurative medikamentöse Therapie gibt. Darüber hinaus ist die PH bislang nur invasiv über eine RHK zu diagnostizieren. Aktuell gibt es in der Forschung das Bestreben, Biomarker zu identifizieren, die die Diagnose einer PH vereinfachen, den Schweregrad der Erkrankung abschätzen lassen oder eine bessere Aussage zum Krankheitsverlauf zulassen. Der Entwicklung einer PH werden Veränderungen im Gefäßsystem zugeschrieben, im Wesentlichen eine Mediahypertrophie, proliferative und fibrotische Veränderungen der Intima sowie inflammatorisch perivaskuläre Infiltrationen.Hypothese: Ang-1 und Ang-2 sind Proteine, die sowohl bei der physiologischen Entwicklung stabiler Gefäße beteiligt sind, aber auch im Zusammenhang mit Remodelling, EC-Proliferation und inflammatorischen Vorgängen genannt werden. Wir überprüften, ob signifikant erhöhte Konzentrationen der Proteine bei Patienten mit PH zu finden sind und ob sie diagnostisch, prognostisch oder zu Abschätzung des Schweregrades geeignet sind.Methoden: Plasma von 151 Patienten bei Ang-1 und 137 Patienten bei Ang-2 wurde im Labor auf die Konzentration von Ang-1 und Ang-2 untersucht und statistisch in Korrelation gesetzt zum 6-MWT und der Hämodynamik der Patienten. Weiterhin wurden die Überlebensstatistiken des Kollektivs ausgewertet. Verglichen wurde es mit Plasma von 35 Kontroll-Patienten, bei denen eine PH mittels RHK ausgeschlossen wurde.Ergebnisse: Bei Ang-1 fanden wir bei keiner der Ätiologien eine signifikante Erhöhung der Konzentration und keine Korrelation mit den Leistungs- oder Hämodynamikparametern. Es gab auch keine Korrelation mit dem Überleben der Patienten. Ang-2 hingegen war im Vergleich zu den Kontrollen signifikant erhöht bei Patienten mit CVD und PVH. Weiterhin fanden wir Korrelationen mit der Hämodynamik bei Patienten mit einer IPAH, CVD und CTEPH.Fazit: Ang-1 zeigte sich in unserer Studie ohne diagnostisches oder prognostisches Potential. Ang-2 könnte bei der Diagnose einer CVD oder PVH eine Rolle spielen. Zusätzlich scheint es ein guter Parameter zur Abschätzung des Schweregrades einer PH zu sein und ggf. als Verlaufsparameter zur Evaluation des Erfolges einer spezifischen Therapie dienen., Background: PH is a severe and progressive disease for which so far no curative drug therapy is available. In addition the only way to diagnose PH is the RHK. At the moment different scientific groups try to identify biomarkers which might help to diagnose PH, display the severity of the desease or that allow to make better predictions of the outcome. There are various changes in the structur of the vascular system during developement of PH. These are mediahypertrophy, proliferation and fibrotic changes of the intima and inflammatory infiltrations.Hypothesis: Our focus was on two proteins, Ang-1 und Ang-2, known to be important in the developement and stabilisation of human vascular system as well as causing vascular remodeling, EC proliferation and inflammation. We investigated if there are sginificant higher levels of these proteins in blood of patients with PH. Moreover we wanted to know if plasma levels of Ang-1 or Ang-2 have diagnostic or prognostic value or might help evaluating disease severity.Methods: We analized plasma levels of Ang-1 in blood-samples of 151 PH patients and levels of Ang-2 in 137 samples. Then we statisctically searched for correlations with 6-MWT and haemodynamics. In addition we analyzed survival rates of all patients as well as of each subgroup. Blood samples of 35 patients without PH were taken as control.Results: Neither did we find significant higher levels of Ang-1 in blood samples of patients with PH compared to our control nor could we find any correlations between plasma levels of Ang-1 and disease severity or survival rates. Ang-2 on the other hand showed significant higher plasma levels in patients with CVD or PVH. We also found correlations with haemodynamics and therefore with disease severity in patients with IPAH, CVD and CTEPH. Similar to the analysis of Ang-1 we found no significant correlation with survival rates.Conclusion: In our study Ang-1 did not show any diagnostic or pronostic value. Ang-2 might serve as a new marker in diagnosis of CVD or PVH in the future. In addition Ang-2 correlated strongly with other markers of disease severity and might be useful as a marker to evaluate success of specific therapy.

Hintergrund: Die PH ist eine schwerwiegende und fortschreitende Erkrankung, für die es bislang keine kurative medikamentöse Therapie gibt. Darüber hinaus ist die PH bislang nur invasiv über eine RHK zu diagnostizieren. Aktuell gibt es in der Forschung das Bestreben, Biomarker zu identifizieren, die die Diagnose einer PH vereinfachen, den Schweregrad der Erkrankung abschätzen lassen oder eine bessere Aussage zum Krankheitsverlauf zulassen. Der Entwicklung einer PH werden Veränderungen im Gefäßsystem zugeschrieben, im Wesentlichen eine Mediahypertrophie, proliferative und fibrotische Veränderungen der Intima sowie inflammatorisch perivaskuläre Infiltrationen. Hypothese: Ang-1 und Ang-2 sind Proteine, die sowohl bei der physiologischen Entwicklung stabiler Gefäße beteiligt sind, aber auch im Zusammenhang mit Remodelling, EC-Proliferation und inflammatorischen Vorgängen genannt werden. Wir überprüften, ob signifikant erhöhte Konzentrationen der Proteine bei Patienten mit PH zu finden sind und ob sie diagnostisch, prognostisch oder zu Abschätzung des Schweregrades geeignet sind. Methoden: Plasma von 151 Patienten bei Ang-1 und 137 Patienten bei Ang-2 wurde im Labor auf die Konzentration von Ang-1 und Ang-2 untersucht und statistisch in Korrelation gesetzt zum 6-MWT und der Hämodynamik der Patienten. Weiterhin wurden die Überlebensstatistiken des Kollektivs ausgewertet. Verglichen wurde es mit Plasma von 35 Kontroll-Patienten, bei denen eine PH mittels RHK ausgeschlossen wurde. Ergebnisse: Bei Ang-1 fanden wir bei keiner der Ätiologien eine signifikante Erhöhung der Konzentration und keine Korrelation mit den Leistungs- oder Hämodynamikparametern. Es gab auch keine Korrelation mit dem Überleben der Patienten. Ang-2 hingegen war im Vergleich zu den Kontrollen signifikant erhöht bei Patienten mit CVD und PVH. Weiterhin fanden wir Korrelationen mit der Hämodynamik bei Patienten mit einer IPAH, CVD und CTEPH. Fazit: Ang-1 zeigte sich in unserer Studie ohne diagnostisches oder prognostisches Potential. Ang-2 könnte bei der Diagnose einer CVD oder PVH eine Rolle spielen. Zusätzlich scheint es ein guter Parameter zur Abschätzung des Schweregrades einer PH zu sein und ggf. als Verlaufsparameter zur Evaluation des Erfolges einer spezifischen Therapie dienen. Background: PH is a severe and progressive disease for which so far no curative drug therapy is available. In addition the only way to diagnose PH is the RHK. At the moment different scientific groups try to identify biomarkers which might help to diagnose PH, display the severity of the desease or that allow to make better predictions of the outcome. There are various changes in the structur of the vascular system during developement of PH. These are mediahypertrophy, proliferation and fibrotic changes of the intima and inflammatory infiltrations. Hypothesis: Our focus was on two proteins, Ang-1 und Ang-2, known to be important in the developement and stabilisation of human vascular system as well as causing vascular remodeling, EC proliferation and inflammation. We investigated if there are sginificant higher levels of these proteins in blood of patients with PH. Moreover we wanted to know if plasma levels of Ang-1 or Ang-2 have diagnostic or prognostic value or might help evaluating disease severity. Methods: We analized plasma levels of Ang-1 in blood-samples of 151 PH patients and levels of Ang-2 in 137 samples. Then we statisctically searched for correlations with 6-MWT and haemodynamics. In addition we analyzed survival rates of all patients as well as of each subgroup. Blood samples of 35 patients without PH were taken as control. Results: Neither did we find significant higher levels of Ang-1 in blood samples of patients with PH compared to our control nor could we find any correlations between plasma levels of Ang-1 and disease severity or survival rates. Ang-2 on the other hand showed significant higher plasma levels in patients with CVD or PVH. We also found correlations with haemodynamics and therefore with disease severity in patients with IPAH, CVD and CTEPH. Similar to the analysis of Ang-1 we found no significant correlation with survival rates. Conclusion: In our study Ang-1 did not show any diagnostic or pronostic value. Ang-2 might serve as a new marker in diagnosis of CVD or PVH in the future. In addition Ang-2 correlated strongly with other markers of disease severity and might be useful as a marker to evaluate success of specific therapy.

Diabetes is a complex metabolic disorder characterized by the failure to maintain normoglycemia stemming from dysfunctional islet of Langerhans. It is caused by an autoimmune destruction of insulin secreting beta-cells in case of type 1 diabetes (T1D), or non-insulin dependent diabetes, caused by lack on insulin action and production in type 2 diabetes (T2D) and by insulin insufficiency during pregnancy as in gestational diabetes mellitus (GDM). T2D accounts for at least 90% of the cases of diabetes, although it may remain undetected or at a pre-diabetic stage for several years. Thus, therapeutic intervention to prevent the progression to T2D is a major goal to subside the incidence of the disease and thus prevent further metabolic complications. T2D is most commonly associated with obesity and thus peripheral insulin resistance. In the face of insulin resistance there occurs an array of molecular mechanisms, one of the major activator being inflammation. In serum, adipose tissue, liver and pancreatic islets from T2D patients, pro-inflammatory cytokines like IL-1beta, CXCL10, TNFalpha, IL-6 have been detected and clinical trials are initiated to prevent inflammatory action. In our study, we showed anti-mouse CXCL10 antibody prevented diabetes progression; it improved glucose tolerance, insulin sensitivity and restored glucose stimulated insulin secretion in the HFD fed mice. CXCL10 antagonism also prevented upregulation of pro-inflammatory cytokines, IL-1beta, IL-6 and CXCL10 mRNA in isolated islets, CXCL10 in adipose tissue and liver of high fat/high sucrose diet (HFD) fed mice. Dipeptidyl peptidase-4 (DPP-4) inhibitors are oral antidiabetics widely in use for T2D treatment. The first agent of its class sitagliptin was approved by the FDA in 2006 and since has been investigated for its direct effects on islet function. The gluco-incretin hormones GIP and GLP-1 secreted by the intestinal endocrine cells potentiate glucose stimulated insulin secretion but are rapidly inactivated by DPP-4. We treated cultured human islets with a diabetic milieu of high glucose, palmitate, cytokines and H2O2 in presence of the DPP-4 inhibitor linagliptin. Linagliptin restored beta-cell function and turnover, via mechanism involving stabilization of secreted GLP-1 in islet supernatants. Obesity induced insulin resistance requires expansion of beta-cell mass to maintain normoglycemia. There occurs a compensatory islet hyperplasia, progressing with altered islet vascularization and possibly angiogenesis, inflammation and eventually leading to reduced beta-cell mass, beta-cell failure and hyperglycemia. The molecular mechanisms involved in the concomitant pathophysiology of islet endothelial cells in T2D has focused on effects mediated by VEGF-A. In this study, we aimed to identify the regulation of and the changes driven by the Angiopoietin/Tie angiogenic factors in islet vascularization and function during the progression of T2D. Ang-1 expressed by perivascular cells and beta-cells and Ang-2 expressed by endothelial cells exert their autocrine and paracrine effects via the cognate receptor Tie-2, on the endothelial cells. Tie-2 signaling maintains quiescent vasculature via constitutive Ang-1 expression whereas Ang-2 is known to be in play in demand for angiogenesis or pathological stimuli involving inflammation. Ang/Tie regulation and thus its role in diabetes and islet vascularization is so far poorly understood. Islet vessel area was increased in autopsy pancreases from T2D subjects, compared to controls. Vessel markers Tie-1, Tie-2 and CD31 were upregulated in mouse islets upon HFD feeding from 8 to 24 weeks. Ang-2 was transiently upregulated in mouse islets at 8 weeks of HFD as well under gluco-lipotoxicity in vitro in human and mouse islets, in contrast to its downregulation with cytokine treatment. Ang-1 on the other hand was oppositely regulated, with reduction under glucolipotoxic conditions and upregulation by cytokine milieu. Modulation of such changes in Ang-2 expression by its overexpression or the inhibition of its receptor Tie-2 impaired beta-cell function at basal conditions but protected islets from cytokine induced apoptosis in vitro. In vivo, beta-cell-specific Ang-2 overexpression in mice induced vascularization under normal diet but contrastingly hypovascularized islets under HFD together with increased apoptosis and reduction of beta-cell mass. Our data show that increased islet hypervascularization is paralleled with T2D. Maintaining physiological Ang-2 levels is important for islet vascularization and beta-cell survival.

Mestrado em Biomedicina Molecular Angiogenesis is crucial for breast cancer growth, progression and dissemination, making it a promising therapeutic target. Given the dominant role of Vascular Endothelial Growth Factor (VEGF) signaling in tumor angiogenesis, the majority of clinical studies with anti-angiogenic therapies in breast cancer were based on VEGF blockade. However, lack of clinical efficacy was observed in all clinical studies with antiangiogenic therapies for breast cancer. Since angiopoietin 2 (ANG-2) has been reported to induce therapeutic resistance after VEGF-targeted therapies, breast cancer patients may benefit from a therapeutic strategy that simultaneously blocks ANG-2 and VEGF-A. Indeed, using the bispecific ANG-2/VEGF-A targeting antibody (A2V), durable inhibition of tumor progression, as well as increased survival, could be observed after dual ANG-2/VEGF-A blockade in the aggressive mouse model of breast cancer, the MMTV-PyMT transgenic mouse. Consistent with tumor inhibition, increased tumor necrosis and a drastic reduction of vascular density was observed after A2V therapy, with little evidence of tumor revascularization or resistance to therapy. Moreover, A2V led to increased perivascular coverage of the remaining tumor blood vessels, favoring vascular normalization, which is known to facilitate the development of an immunosupportive microenvironment. These findings suggested that breast cancer immunotherapy may benefit from the combination with A2V. Therefore the A2V mAb was combined with a CD40 agonistic antibody (FGK45). The combined therapeutic approach led to remarkable tumor growth inhibition in an orthotopic MMTV-PyMT model, with the achievement of cases of stable disease and further increase in tumor necrosis. Increased infiltration of CD8+ cells after FGK45 therapy, either alone or in combination with A2V, could also be observed. Of note, a correlation between the extent of tumor inhibition and increased tumor infiltration by CD8+ cells was detected, suggesting that the influx of CD8+ cells in the combination therapy-treated tumors was responsible for the remarkable anti-tumor responses observed. In summary, these results point to a significant synergistic activity of anti-ANG-2/VEGF-A and CD40 agonistic therapy in the MMTV-PyMT model of breast carcinoma. This combinatorial approach may benefit breast cancer patients, so further studies that explore the mechanistic bases of the cooperative activity of antiangiogenic and immunostimulatory therapy are warranted. A angiogénese é um processo crucial para o crescimento, progressão e disseminação do cancro da mama, tornando-se assim um alvo terapêutico promissor. A maioria dos estudos clínicos de terapias anti-angiogénicas em cancro da mama baseiam-se na neutralização do fator de crescimento endotelial vascular (VEGF), uma vez que este apresenta um papel dominante na angiogénese tumoral. No entanto, todos os estudos clínicos baseados em terapias anti-angiogénicas têm-se revelado ineficazes no tratamento de pacientes com cancro da mama. Uma vez que a Angiopoietina 2 (ANG-2), outro fator de crescimento vascular, tem sido reportada como responsável pela resistência a terapias baseadas no bloqueio de função do VEGF, é possível que pacientes com cancro da mama beneficiem de uma estratégia terapêutica que neutralize simultaneamente ANG-2 e VEGF-A. De facto, através do uso de um anticorpo ‘biespecífico’, capaz de neutralizar tanto ANG-2 como VEGF-A e denominado A2V, observou-se uma forte inibição da progressão tumoral e aumento da sobrevivência num modelo pré-clínico de cancro da mama agressivo, baseado em ratinhos transgénicos e denominado MMTV-PyMT. Consistentemente, esta terapia levou ao aumento de necrose tumoral e a uma drástica redução na densidade vascular, sem evidência de revascularização ou resistência à terapia. A administração de A2V induziu ainda o aumento da associação entre células perivasculares (‘pericytes’) e células endoteliais dos restantes vasos sanguíneos tumorais, favorecendo a maturação e normalização vascular, o que facilita o desenvolvimento de um microambiente que suporta respostas imunológicas. Estes resultados sugeriam assim que imunoterapia em cancro da mama poderia beneficiar da combinação com A2V, tendo-se por conseguinte combinado o anticorpo A2V com o anticorpo agonista de CD40 (FGK45), no modelo ortotópico de MMTV-PyMT. Esta terapia induziu uma notável inibição do crescimento tumoral, com obtenção de casos estáveis, e aumento adicional de necrose tumoral. Observou-se ainda o aumento de infiltração de células CD8+ após terapia com FGK45, quer isoladamente ou em combinação com A2V, e uma correlação entre a extensão da inibição do tumor e o aumento da infiltração tumoral por células CD8+. Isto sugere que o influxo de células CD8+ nos tumores tratados com A2V e FGK45 foi o principal responsável pelas notáveis respostas anti-tumorais observadas. Em resumo, estes resultados apontam para uma sinergia entre a neutralização simultânea de ANG-2/VEGF-A e imunoterapia baseada em agonistas do recetor CD40, no modelo pré-clínico de cancro da mama MMTV-PyMT. Esta abordagem combinatória pode beneficiar pacientes com cancro da mama, por isso, mais estudos são necessários para explorar os mecanismos subjacentes à atividade cooperativa da terapia anti-angiogénica e a imunoterapia.

9 p.-4 fig., Hereditary hemorrhagic telangiectasia (HHT) is a genetically heterogeneous disorder, involving mutations in two predominant genes known as Endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2), as well as in some less frequent genes, such as MADH4/SMAD4 (JP-HHT) or BMP9/GDF2 (HHT5). The diagnosis of HHT patients currently remains at the clinical level, according to the “Curaçao criteria,” whereas the molecular diagnosis is used to confirm or rule out suspected HHT cases, especially when a well characterized index case is present in the family or in an isolated population. Unfortunately, many suspected patients do not present a clear HHT diagnosis or do not show pathogenic mutations in HHT genes, prompting the need to investigate additional biomarkers of the disease. Here, several HHT biomarkers and novel methodological approaches developed during the last years will be reviewed. On one hand, products detected in plasma or serum samples: soluble proteins (vascular endothelial growth factor, transforming growth factor b1, soluble endoglin, angiopoietin-2)and microRNA variants (miR-27a, miR-205, miR-210). On the other hand, differential HHT gene expression fingerprinting, next generation sequencing of a panel of genes involved in HHT, and infrared spectroscopy combined with artificial neural network patterns will also be reviewed. All these biomarkers might help to improve and refine HHT diagnosis by distinguishing from the non-HHT population., This study has been supported by grants from Ministerio de Economia y Competitividad of Spain(SAF2011-23475toLMB;SAF2013-43421-R andSAF2010-19222toCB)and Centro de Investigación Biomedica en Red de Enfermedades Raras (CIBERER).CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain,supported by FEDER funds.

Angiogenesis is a complex process involving dynamic interaction of various cell to cell interactions. Endothelial cell interactions regulated by growth factors, inflammatory cytokines, or hemodynamic stress are critical for balancing vascular quiescence and activation. Yes-associated protein (YAP), an effector of Hippo signaling, is known to play significant roles in maintaining cellular homeostasis. However, its role in endothelial cells for angiogenic regulation remains relatively unexplored. We demonstrated the critical role of YAP in vascular endothelial cells and elucidated the underlying molecular mechanisms involved in angiogenic regulation of YAP. YAP was expressed in active angiogenic regions where endothelial cell junctions were relatively loosened. Consistently, YAP subcellular localization and activity were regulated by VE-cadherin-mediated PI3K/Akt pathway. YAP thereby regulated endothelial sprouting via angiopoietin-2 expression. These results provide an insight into a model of coordinating endothelial junctional stability and angiogenic activation through YAP. [BMB Reports 2015; 48(8): 429-430]

Type 2 diabetes is the most common metabolic disorder today and has reached epidemic proportions in many countries. Diet-induced insulin resistance plays a central role in the development of type 2 diabetes. Studies included in this thesis describe findings regarding two forkhead genes, Foxf2 and Foxc2, and their involvement in the development of insulin resistance. We produced a mouse in which the forkhead factor FOXF2 is overexpressed in an adipose tissue-restricted fashion, such mice display induced insulin secretion in response to an intravenous glucose load. In addition we could demonstrate that adipocytes from FOXF2 transgenic mice have an impaired insulin-mediated glucose uptake. We argue this to be, at least in part, due to lower expression of insulin receptor substrate 1. Mice overexpressing forkhead factor FOXC2 in adipose tissue have previously been shown to be protected from diet-induced obesity and glucose intolerance. In hyperinsulinemic-euglycemic clamp experiments, we demonstrated that FOXC2 transgenic mice are protected from diet-induced insulin resistance in liver and skeletal muscle. Furthermore, on high-fat diet, FOXC2 transgenic mice displayed decreased intramuscular levels of fatty acyl CoA compared with wild-type littermates. Expansion and regression of adipose tissue requires continuous remodelling of the vasculature in order to meet demands of metabolism. We have shown that the adipose tissue of FOXC2 transgenic mice exhibit a higher vascular density and altered pattering of vascular smooth muscle cells and pericytes. Furthermore, we could show this, at least in part, to be dependent on the role of FOXC2 as a direct regulator of Angiopoietin 2.

Argument: Hypertension is a main contributing risk factor to many cardiovascular diseases and may be the cause or the result of cardiovascular dysfunction. Black Africans, especially, suffer from hypertension because of lifestyle changes that occur during westernisation, which may lead to sympatho-adrenal hyperactivity. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are regulators of angiogenesis and are significantly up regulated during states of vascular dysfunction. Levels of angiogenic factors are unknown for African people and may not be the same as levels thus far reported for Caucasians. Aims: The aim of this study is firstly, to determine whether differences exist regarding the levels of VEGF-A and Ang-2 in urbanised compared to rural black Africans and secondly, to determine whether increased levels of VEGF-A and Ang-2 factors are related to hypertension in black Africans. Methodology: This is a sub study that is based upon the Prospective Urban and Rural Epidemiological (PURE) study. Apparently healthy, fasting African men and women (N=272, aged 35 to 50 years) from the North-West province of South Africa were selected by a medical doctor to participate in this study. Groups were stratified according to gender and urbanisation status based upon information derived from sociodemographic questionnaires. Cardiovascular parameters (Omron HEM-757), pulse wave velocity (PWV) (Compiler SP), plasma angiogenic factor levels (ELISA) and anthropometric measures were determined. An independent t-test and Pearson Chi-square test were used to compare urban and rural data, followed by an analysis of covariance (ANCOVA) while correcting for confounders (age, body mass index, physical activity and tobacco usage). ANCOVAs (corrected for confounders) were applied where hypertensive and normotensive groups were compared within the whole group and urbanised groups. Correlations, correcting for confounders, between cardiovascular variables and angiogenic factors were determined within the whole group and urbanised groups. Results and conclusion: Plasma VEGF-A values for all black Africans were very low while the ANG-2 levels were elevated compared to control values for Caucasians (normotensive and hypertensive) in literature. Urbanised men were more overweight and indicated a higher incidence of hypertension (42.47%) and elevated VEGF-A levels, but lower Ang-2 levels compared to rural men. Urbanised women were generally overweight, physically less active and smoked less, but indicated higher diastolic blood pressure (BP), VEGF-A levels and lower PWV compared with their rural counterparts. Ang-2 levels indicate a negative relationship to diastolic BP data in rural women. No relationships between hypertensive individuals and high angiogenic factor levels were uncovered. Conclusive evidence suggested that angiogenic factor levels were affected more by urbanisation than by the state of hypertension. If low levels of VEGF-2 occur, ANG-2 stimulation and properties may be altered, thereby switching ANG-2 from an anti-angiogenic to a pro-angiogenic molecule, inferring blood vessel destabilisation and vascular dysfunction, such as is observed in hypertensive urbanised men. Higher ANG-2 levels may result in Tie-2 receptor down regulation, hence causing VEGF-A levels to be lower. Further study is needed to ascertain this mechanism since Tie-2 receptor activity was not determined in this study. Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2009.

Thesis (M.Sc. (Physiology))--North-West University, Potchefstroom Campus, 2009. Argument: Hypertension is a main contributing risk factor to many cardiovascular diseases and may be the cause or the result of cardiovascular dysfunction. Black Africans, especially, suffer from hypertension because of lifestyle changes that occur during westernisation, which may lead to sympatho-adrenal hyperactivity. Vascular endothelial growth factor-A (VEGF-A) and angiopoietin-2 (Ang-2) are regulators of angiogenesis and are significantly up regulated during states of vascular dysfunction. Levels of angiogenic factors are unknown for African people and may not be the same as levels thus far reported for Caucasians. Aims: The aim of this study is firstly, to determine whether differences exist regarding the levels of VEGF-A and Ang-2 in urbanised compared to rural black Africans and secondly, to determine whether increased levels of VEGF-A and Ang-2 factors are related to hypertension in black Africans. Methodology: This is a sub study that is based upon the Prospective Urban and Rural Epidemiological (PURE) study. Apparently healthy, fasting African men and women (N=272, aged 35 to 50 years) from the North-West province of South Africa were selected by a medical doctor to participate in this study. Groups were stratified according to gender and urbanisation status based upon information derived from sociodemographic questionnaires. Cardiovascular parameters (Omron HEM-757), pulse wave velocity (PWV) (Compiler SP), plasma angiogenic factor levels (ELISA) and anthropometric measures were determined. An independent t-test and Pearson Chi-square test were used to compare urban and rural data, followed by an analysis of covariance (ANCOVA) while correcting for confounders (age, body mass index, physical activity and tobacco usage). ANCOVAs (corrected for confounders) were applied where hypertensive and normotensive groups were compared within the whole group and urbanised groups. Correlations, correcting for confounders, between cardiovascular variables and angiogenic factors were determined within the whole group and urbanised groups. Results and conclusion: Plasma VEGF-A values for all black Africans were very low while the ANG-2 levels were elevated compared to control values for Caucasians (normotensive and hypertensive) in literature. Urbanised men were more overweight and indicated a higher incidence of hypertension (42.47%) and elevated VEGF-A levels, but lower Ang-2 levels compared to rural men. Urbanised women were generally overweight, physically less active and smoked less, but indicated higher diastolic blood pressure (BP), VEGF-A levels and lower PWV compared with their rural counterparts. Ang-2 levels indicate a negative relationship to diastolic BP data in rural women. No relationships between hypertensive individuals and high angiogenic factor levels were uncovered. Conclusive evidence suggested that angiogenic factor levels were affected more by urbanisation than by the state of hypertension. If low levels of VEGF-2 occur, ANG-2 stimulation and properties may be altered, thereby switching ANG-2 from an anti-angiogenic to a pro-angiogenic molecule, inferring blood vessel destabilisation and vascular dysfunction, such as is observed in hypertensive urbanised men. Higher ANG-2 levels may result in Tie-2 receptor down regulation, hence causing VEGF-A levels to be lower. Further study is needed to ascertain this mechanism since Tie-2 receptor activity was not determined in this study. Masters

International audience; Sodium (Na+) ions are known to regulate many signaling pathways involved in both physiological and pathological conditions. In particular, alterations in intracellular concentrations of Na+ and corresponding changes in membrane potential are known to be major actors of cancer progression to metastatic phenotype. Though the functionality of Na+ channels and the corresponding Na+ currents can be investigated using the patch-clamp technique, the latter is rather invasive and a technically difficult method to study intracellular Na+ transients compared to Na+ fluorescence imaging. Despite the fact that Na+ signaling is considered an important controller of cancer progression, only few data using Na+ imaging approaches are available so far, suggesting the persisting challenge within the scientific community. In this study, we describe in detail the approach for application of Na+ imaging technique to measure intracellular Na+ variations in human prostate cancer cells. Accordingly, we used three Na+-specific fluorescent dyes-Na+-binding benzofuran isophthalate (SBFI), CoroNa™ Green (Corona) and Asante NaTRIUM Green-2 (ANG-2). These dyes have been assessed for optimal loading conditions, dissociation constant and working range after different calibration methods, and intracellular Na+ sensitivity, in order to determine which probe can be considered as the most reliable to visualize Na+ fluctuations in vitro.

Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm2, 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

Background Angiopoietin-2 (Ang-2) plays critical roles in vascular morphogenesis and its upregulation is frequently associated with various tumors. Previous studies showed that certain selenium compounds possess anti-tumor effects. However, the underlining mechanism has not been elucidated in detail. Plus, results of research on the anti-tumor effects of selenium compounds remain controversial. Methods We investigated levels of Ang-2 and vascular endothelial growth factor (VEGF) on the estrogen-independent bone metastatic mammary cancer (MDA-MB-231) cells in response to treatment by methylseleninic acid (MSeA), and further examined the effects of MSeA oral administration on xenograft mammary tumors of athymic nude mice by RT-PCR, Western, radioimmuno assay, and Immunohistochemistry. Results Treatment of MDA-MB-231 cells with MSeA caused significant reduction of Ang-2 mRNA transcripts and secretion of Ang-2 proteins by the cells. Level of VEGF protein was accordingly decreased following the treatment. Compared with the controls, oral administration of MSeA (3 mg/kg/day for 18 days) to the nude mice carrying MDA-MB-231 induced tumors resulted in significant reduction in xenograft tumor volume and weights, significant decrease in microvascular density, and promotion of vascular normalization by increasing pericytes coverage. As expected, level of VEGF was also decreased in MSeA treated tumors. Conclusions Our results point out that MSeA exerts its anti-tumor effects, at least in part, by inhibiting the Ang-2/Tie2 pathway, probably via inhibiting VEGF.

Background Pancreatic cancer is currently one of the leading causes of cancer deaths without any effective therapies. Mir-145 has been found to be tumor-suppressive in various types of cancers. The aim of this study is to investigate the role of miR-145 in pancreatic cancer cells and explore its underlying mechanism. Methods Quantitative real time PCR was used to determine the expression level of miR-145 and angiopoietin-2 (Ang-2) mNRA, and the expression level of Ang-2 protein was measured by western blotting. The anti-cancer activities of miR-145 were tested both in in vitro by using cell invasion and colony formation assay and in vivo by using xenograft assay. The direct action of miR-145 on Ang-2 was predicted by TargetScan and confirmed by luciferase report assay. The vascularization of xenografts were performed by immunohistochemical analysis. Results The expression level of miR-145 was significantly lower and the expression levels of Ang-2 mRNA and protein was significantly higher in the more aggressive pancreatic cancer cells (MiaPaCa-2 and Panc-1) when compared to that in BxPC3 cells. Overexpression of miR-145 in the BxPC3, MiaPaCa-2 and Panc-1 cells suppressed the cell invasion and colony formation ability, and the expression level of Ang-2 protein in MiaPaCa-2 and Panc-1 cells was also suppressed after pre-miR-145 transfection. Intratumoral delivery of miR-145 inhibited the growth of pancreatic cancer xenografts and angiogenesis in vivo, and also suppressed the expression level of angiopoietin-2 protein. Luciferase report assay showed that Ang-2 is a direct target of miR-145, and down-regulation of angiopoietin-2 by treatment with Ang-2 siRNA in the BxPC3, MiaPaCa-2 and Panc-1 cells suppressed cell invasion and colony formation ability. The reverse transcription PCR results also showed that Tie1 and Tie2 were expressed in BxPC3, MiaPaCa-2 and Panc-1 cells. Conclusion MiR-145 functions as a tumor suppressor in pancreatic cancer cells by targeting Ang-2 for translation repression and thus suppresses pancreatic cancer cell invasion and growth, which suggests that restoring of miR-145 may be a potential therapeutic target for pancreatic cancer.