Angiogenesis is a process of new blood vessels sprouting from the pre-existing vasculature, and mediated by multiple angiogenic and anti-angiogenic factors. Disturbance of the balance often leads to development of neovascular diseases. Neovascularization affecting the eye is a common cause of visual impairment and even blindness, particularly when corneal or choroidal neovascularization (NV) is involved. While there are effective treatment modes for ocular neovascularization, they are expensive and only inhibit disease progress. Since herbal medicine has been applied for anti-angiogenesis and anti-carcinogenesis therapies, we investigate the anti-angiogenic effect of selected herbal molecules: isoliquiritigenin (ISL), a flavonoid from licorice; epigallocatechin gallate (EGCG), a polyphenol from green tea; and resveratrol (Rst), a polyphenol phytoalexin derived from grapes. / In conclusion, by in vitro and in vivo studies, we showed that ISL, EGCG and Rst contributed to anti-angiogenesis via different biological mechanisms. We propose that these three herbal molecules (ISL, EGCG and Rst) are candidate anti-angiogenic agents for the treatment of ocular angiogenesis diseases. Their distribution profiles and pharmacokinetic properties should be investigated. / Results showed that sub-toxic levels of ISL (10 microM), EGCG (50 microM) and Rst (10microM) effectively suppressed endothelial cell proliferation and migration in the scratch-wound assay. Treatment with ISL was found to significantly up-regulate PEDF, which is known as a potent angiostatic factor. EGCG and Rst downregulate VEGF signaling cascade by suppressing Akt and FAK activation and affecting MMP-2, MMP-9 expression. In vivo angiogenesis assays further showed the suppressive effect of ISL, EGCG and Rst on neovascularization in three different animal models. Application of ISL at 1 microM showed the suppressive effect on chick CAM assay, corneal NV and choroidal NV assays consistently, the most effective dosage was close to 10 microM. EGCG at 1 microM showed the effect to reduce chick CAM vessel formation and corneal NV, and at 10 microM (the lowest tested concentration) to suppress choroidal NV in mice. Variable effects were observed in Rst treatment. Rst at 10 microM prohibited vessel growth in chick CAM, and 1 microM suppressed corneal NV formation and 2 microM deterred choroidal NV development. / This thesis contains two major parts. The first in vitro cell-based analysis investigated the toxicity of these herbal chemicals and their effect on endothelial cell growth and migration. The expression profile of vascular endothelial growth factor (VEGF) signaling cascade events, including Akt and focal adhesion kinase (FAK) activation, VEGF, pigment epithelium-derived factor (PEDF) and matrix metalloproteinases (MMPs) were examined by Western blotting. Then three in vivo models were established to study the effect of these herbal chemicals on angiogenesis. They were (1) developmental angiogenesis in chick chorioallantoic membrane (CAM), (2) pathological angiogenesis in silver nitrate cauterization-induced corneal neovascularization in BALB/c mice and, (3) laser photocoagulation-induced choroidal neovascularization in C57BL/6 mice. Changes of vascularization were determined by qualification of vessel number changes on the edge of gelatin sponge in 24 hours (chick CAM assay), measurement of vascularized area, live imaging of vessel leakage (fundus fluorescence angiography, FFA) and immunochemistry using antibodies specific for endothelial cells (corneal & choroidal NV assays) respectively. / Liu, Huanming. / Adviser: Chi Pui Pang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 155-180). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Lorsque des vaisseaux autologues ne sont pas disponibles pour faire un pontage, des greffons synthétiques sont utilisés mais avec des taux d’échec élevés. En effet, malgré leurs bonnes propriétés mécaniques, la surface synthétique de ces greffons entraîne de la thrombose et de l’hyperplasie intimale ayant pour conséquence une mauvaise perméabilité du substitut à long terme pour de nombreuses applications. Par ingénierie tissulaire, des greffons vasculaires entièrement biologiques et humains ont déjà été produits par roulage de feuillets de matrice extracellulaire synthétisée par des fibroblastes dermiques humains in vitro. Grâce à une nouvelle méthode d’assemblage basée sur une approche textile, des greffons ont été produits trois fois plus rapidement. Pour ce faire, le feuillet a été découpé en fils afin de permettre la construction d’un substitut vasculaire par tissage. Cette thèse comporte trois articles. Le premier visait à montrer la composition riche de la matrice, décrire l’organisation de son réseau complexe de collagènes et démontrer que la dévitalisation par séchage de la matrice n’a pas affecté significativement cette organisation. Le deuxième avait pour but de décrire les propriétés mécaniques des fils en fonction du torsadage et/ou de l’âge de la matrice ainsi que l’effet sur la force de différents traitements nécessaires au processus de fabrication. Les différentes applications de l’approche textile dans la construction de structures complexes ainsi que les propriétés mécaniques des substituts tissés ont également été évaluées. Le troisième article a montré la faible réponse inflammatoire ainsi que le potentiel d’intégration et de remodelage de la matrice in vivo. Par ailleurs, la décellularisation n’a pas montré de résultats supérieurs à la dévitalisation, permettant ainsi de s’affranchir d’une étape de fabrication supplémentaire et potentiellement délétère à l’organisation biologique de cette matrice. En conclusion, cette thèse constitue la première démonstration de la fabrication de textiles humains mécaniquement très forts mais sans utilisation de matériel exogène. La dévitalisation couplée à l’approche textile ont permis de créer un modèle allogénique plus simple, plus rapide et moins coûteux mais avec un potentiel d’intégration in vivo intact. Ce modèle sera très prochainement étudié par implantation à long terme dans la circulation sanguine. / When autologous blood vessels are not available for bypass surgery, synthetic grafts are used but display high failure rates. Indeed, despite their good mechanical properties, their synthetic surface lead to thrombosis and intimal hyperplasia, which cause poor long-term patency in many applications. Using tissue engineering, completely biological and human vascular grafts have been produced by rolling sheets of extracellular matrix synthesized by dermal human fibroblasts in vitro. Using a new assembly technique based on a textile approach, grafts were produced three-time faster. To do so, sheets were cut into yarns to construct vascular substitute by weaving. This manuscript includes three articles. The first one aimed at showing the rich composition of the matrix, describing the organization of its complex network of collagens and demonstrating that the devitalization by drying the matrix did not significantly affect this organization. The second one described the mechanical properties of the yarns depending on the twisting, matrix age or different treatments useful for the manufacturing process. It also demonstrated some of the assembly techniques possible with this human yarn, as well as its possible use as a suture or to build a vascular graft. The third article showed the survival of the yarns subcutaneously implanted for 6 month in nude rats. The implants created little inflammatory response, were mildly remodeled and kept a significant mechanical strength. Decellularization did not show results improvement compared to the simple devitalization, demonstrating that the remaining cellular fragments were not a meaningful activator of the innate immune system. To conclude, this thesis is the first demonstration of the production of human textiles, without using any exogenous material and that are mechanically very strong. Both the devitalization and the textile approach have allowed to create a simpler allogeneic model, faster and cheaper but with an intact potential of integration in vivo, that will be studied very soon with a long-term implantation of the textile in the bloodstream.

Introdução: Até o presente momento, há escassez de estudos que avaliem os níveis séricos de citocinas angiogênicas em pacientes com dermatomiosite (DM), uma miosite autoimune sistêmica que tem como fisiopatogênese a vasculopatia. Portanto, os objetivos do presente estudo foram: (a) analisar sistematica e simultaneamente os níveis séricos de angiogenin (ANG), angiopoietin (ANGPT) -1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) -1 e -2 e platelet derived growth factor (PDGF) -AA e -BB em pacientes com DM; (b) correlacionar o nível sérico destas citocinas com as características clínico-laboratoriais, alterações metabólicas e atividade da DM. Pacientes e métodos: Estudo transversal, único centro, no qual foram incluídos, no período de 2012 a 2014, 48 pacientes consecutivos com DM definida (Bohan e Peter, 1975), entre 18 e 45 anos de idade, e em seguimento regular na nossa Instituição. Foram excluídos pacientes com condições clínicas relacionadas à DM (como sequelas de doença e tratamento prolongado) ou secundárias a outras causas que poderiam influenciar na interpretação dos resultados das citocinas avaliadas, seja por apresentarem variações hormonais ou por estarem relacionados ao mecanismo de inflamação e/ou angiogênese. Os pacientes foram pareados por sexo, idade e etnia com 48 indivíduos saudáveis (grupo controle). A análise das citocinas séricas foi realizada por imunoensaio multiplex. Os parâmetros da atividade da DM foram baseados nos escores estabelecidos por International Myositis Assessment & Clinical Studies Group (IMACS). Resultados: A distribuição de sexo e a etnia foram comparáveis entre os grupos DM e controle assim como a média de idade (33,3±7,6 vs. 35,8±8,2 anos, respectivamente), e a mediana de duração de doença foi de 1 ano. No grupo DM, os níveis séricos de FGF-1 e FGF-2 (P < 0,001, P < 0,001, respectivamente) estavam elevados, enquanto os níveis de VEGF e PDGF-AA (P=0,009 e P=0,022, respectivamente) estavam reduzidos. Os níveis de ANG, ANGPT-1 e PDGFBB foram semelhantes em ambos os grupos. Houve uma tendência para correlação positiva entre as citocinas (com exceção de VEGF e PDGF-BB) e os parâmetros de atividade da DM, enquanto FGF-2 apresentou correlação negativa. Além disso, o FGF-1 correlacionou-se fortemente com manifestações cutâneas da dermatomiosite. Conclusões: Os dados atuais reforçam a importância das citocinas angiogênicas nos mecanismos de vasculopatia da DM, especialmente em condições de atividade cutânea e adequado tratamento medicamentoso. Estudos adicionais serão necessários para validar os dados obtidos no presente trabalho / Introduction: Until now, there are few studies evaluating serum levels of angiogenic cytokines in patients with dermatomyositis (DM), a systemic autoimmune myositis that has vasculopathy as its physiopathogenesis. Therefore, the aims of the present study were: (a) to analyze systematically and simultaneously serum levels of angiogenin (ANG), angiopoietin (ANGPT) -1, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) -1 and -2, and platelet derived growth factor (PDGF) -AA and -BB in patients with DM; (b) to correlate the serum level of these cytokines with the clinical and laboratory features, metabolic alterations and DM activity. Patients and methods: This is an one-center cross sectional study, in which 48 consecutive patients with defined DM (Bohan and Peter, 1975) aged 18 to 45 years and regularly followed up at our Institution were included from 2012 to 2014. Patients with clinical conditions related to DM (as sequelae of disease and prolonged treatment) or secondary to other causes that could influence the results interpretation of the evaluated cytokines were excluded, either because of hormonal variations or relationship with inflammation mechanism and/or angiogenesis. Patients were gender-, age- and ethnicity-matched with 48 healthy individuals (control group). The serum levels of cytokines analyses were performed by multiplex immunoassay. The parameters of DM activity were based on the scores established by the International Myositis Assessment & Clinical Studies Group (IMACS). Results: The gender and ethnicity were comparable between DM and control groups as soon as the mean age (33.3±7.6 vs. 35.8±8.2 years, respectively), and the median disease duration was 1 year. The serum levels of FGF-1 and FGF-2 (P < 0.001 and P < 0.001, respectively) were higher in DM group, whereas the levels of VEGF and PDGF-AA (P=0.009 and P=0.022, respectively) were lower in DM group. The levels of ANG, ANGPT-1 and PDGF-BB were similar in both groups. There was a tendency for cytokines (with the exceptions of VEGF and PDGF-BB) to correlate positively with DM activity parameters, whereas FGF-2 correlated inversely. Moreover, FGF-1 strongly correlated with the cutaneous manifestations of DM. Conclusion: The current data reinforce the importance of angiogenic cytokines in DM vasculopathy mechanisms, especially in conditions of cutaneous activity and adequate drug treatment. Additional studies will be needed to validate the data obtained in this work

Objetivos: Avaliar as características do canal da artéria alveolar superior posterior (AASP) em exames de tomografia de feixe conico (TCFC) em indivíduos com fissura labiopalatina (FI) e comparar com indivíduos não fissurados (NF). Material e Métodos: Foram analisados 150 exames de TCFC de FI e 150 TCFC de NF. Os critérios de exclusão foram: exames de pacientes sindrômicos, presença de lesões intraósseas, desdentados totais, outras alterações que interferem a visivilidade do canal da AASP, presença de artefatos, idade abaixo de 20 anos e exames que não incluíam toda a maxila. As características avaliadas foram à presença/ausência, localização em relação ao seio maxilar, diâmetro, distancia em relação ao rebordo alveolar e assoalho do seio maxilar e término presença/ausência de anastomose com a artéria alveolar superior anterior (AASA).Os testes estatísticos aplicados foram Kappa, Dahlberg Fischers, test t. Resultados: O grupo de FI foi constituído de 75 homens e 75 mulheres, com idade média de 29,8 anos. O grupo de NF foi composto por 75 homens 75 mulheres com idade média de 40,3 anos. O Kappa para a concordância da presença e ausência do canal da AASP foi: intra-avaliador 0,8 substancial e inter-avaliador de 0,7 subtancial. O Dahlberg intra-avaliador foi de 0, 75 excelente e inter-avaliador de 0,7 satisfatório. No ponto de ingresso o canal AASP esteve presente em 100% em FI e em 100% em NF, em todos os casos o canal da AASP era bilateral. Os diâmetros, do canal da AASP, foram significativamente diferentes entre os dois grupos, sendo maior para os FI (Test t P < 0,0001). A localização em relação ao ponto de ingresso no seio maxilar foi estatisticamente significante entre ambos os grupos, nos FI a maior frequência foi no terço médio (Fisher\' s P <0,0001) e superior do seio maxilar (Fisher\' s P < 0,0071 lado esquerdo), e os NF apresentaram maior frequência foi no terço inferior (Fisher\' s P < 0,0001). Em relação à distância da AASP ao rebordo alveolar e ao assoalho do seio maxilar, não houve resultados estatisticamente significantes. Em relação aos tipos de términos não houve diferença estatisticamente significante entre os grupos. Conclusões: De acordo com material e métodos realizados, o canal da AASP apresentou maior diâmetro e localização mais superior no seio maxilar em FI quando comparado com NF. Esses resultados podem indicar um maior risco de sangramento durante cirurgias para os portadores de fissura labiopalatina. / Objectives: This study evaluates the characteristics of the posterior superior alveolar artery canal (AASP) in individuals with cleft lip and palate (FI) and compares with individuals without cleft lip and palate (NF). Material and Methods: 150 Cone-Beam Computed Tomography (TCFC) were evaluated from both FI and NF 150 TCFC according to the criteria outlined above. Exclusion criteria were: Sindromic patients, exams with alteration introsseous, edentulous maxilla, artifacts, people below 20 years of age, exams that did not include all maxilla. The characteristics evaluated include presence/absence, location in to maxillary sinus, diameter, distance in relation to the alveolar crest and maxillary sinus floor and presence/absence of anastomosis with the anterior superior alveolar artery canal (AASA). The statistical tests used were Kappa, Dahlberg, Fisher\'s and test t. Results: The group of FI was composed of 75 men and 75 women, with an average age of 29.8. The NF group was composed of 75 men and 75 women with average of age 40.3. In relation to the presence/absence of the AASP canal, result from an evaluation by the intra-rater was 0,8 substancial and inter-rater showed 0.7substantial. O Dahlberg intra-rater was 0.75 excelent and inter-rater 0.7 satisfactory. The prevalence of the AASP canal was100% in the FI group and NF. Regarding the diameters of the AASP canal there were significant differences between the two groups, and was considerably greater for the FI group (Test t p < 0.0001). With respect to location in relation to the point of entry in the maxillary sinus results showed significant statistical difference between both groups, with the NF group possessing higher frequency in the middle third (Fishers p <0.0001) and upper third of maxillary sinus (Fishers p < 0.0071 left side) and NF had higher frequency was in lower third (Fishers P <0.0001). There was no significant statistical difference in relation to the distance of the AASP to alveolar crest and to the floor maxillary sinus. The same was true in relation to the types of terms. Conclusion: According to the material and methods the AASP canal present larger diameter and more premium location in the maxillary sinus in individuals with FI compared with NF. The results indicate a higher risk of bleeding during surgery for patients with cleft lip and palate.

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Atherosclerosis is a vascular degenerative disease leading to progressive thickening in the intima of large and medium sized arteries through the formation of plaque that is very rich with cholesterol. The cholesterol is carried by LDL (low density lipoprotein) particles which pass through the endothelium and accumulate in the intima. The passage of LDL is influenced by wall shear stress which activates physiological responses of the endothelium. However, the causal relationship between the physiological responses and their effect on LDL mass transport is not fully understood. To obtain blood flow patterns in human carotid arteries, a fluid structure interaction (FSI) computational approach is employed, based on the in-vivo arterial geometry constructed from black blood magnetic resonance images (BBMRI) and flow rate boundary conditions obtained from phase contrast images (PC). Wall shear stress (WSS) on the luminal surface is computed, and this variable is related to the formation of leaky junctions, which is a major transendothelial pathway for LDL. A model for the fraction of leaky junction at a surface is incorporated into the overall computational scheme for mass transport, along with pore theory. The theoretical model is applied to images from three human carotid arteries in which the degree of disease ranges from mild to moderate. Maximum mass flux is predicted to be in the downstream region of stenoses where WSS is low, and this result is consistent with the clinical observation of plaque progression downstream of the stenosis. The hypothesis that the majority of LDL enters into the intima through leaky junctions is supported by observation of similar distributions between the pattern of volume flux via leaky junctions and mass flux. These studies suggest that mass flux of LDL can be a predictor to indicate areas with potential for plaque formation and progression in human carotid artery disease.

Neovascularization, or the formation of blood vessels, is important in both normal physiological processes as well as pathophysiological processes. The main players in neovascularization, endothelial cells (EC), are highly influenced by hemodynamic shear stress and this may play an important role in neovascularization. Two typical types of shear stress found in the vascular system are a unidirectional laminar shear stress (LS) found in straight regions and a disturbed, oscillatory shear stress (OS) found at branches or curves. At the cellular level, LS is thought to promote EC quiescence whereas OS is thought to promote EC dysfunction. Oscillatory sheared EC are pro-proliferative, pro-migratory, and secrete growth factors, all functions important in neovascularization. There are several diseases that involve both disturbed shear stress and neovascularization, such as atherosclerosis, aortic valve disease, and occlusive vascular disease. In these pathophysiological scenarios fluid shear stress may provide a driving force for neovascularization. Therefore, we hypothesized that oscillatory shear stress promotes greater neovascularization compared to unidirectional laminar shear stress through the secretion of angiogenic factors, which play a physiological role in neovascularization in vivo. To test this hypothesis, we first performed tubule formation and migration assays, two important functions in neovessel formation. We found that OS promotes greater tubule formation and migration of EC as compared to LS and this was mediated through secreted factors. Using gene and protein array analysis, we identified Angiopoietin-2 (Ang2) as being upregulated by OS compared to LS in EC. We found that inhibiting Ang2 blocked OS-mediated tubule formation and migration and that LS-inhibited tubule formation could be rescued by addition of Ang2. In addition, Ang2 was found to be upregulated at sites of disturbed flow in vivo, implicating a physiological role for Ang2. To further investigate the physiological role of Ang2 in neovascularization, we examined the effects of inhibiting Ang2 in a mouse model of hindlimb ischemia, which involves both disturbed flow and neovascularization. We found that Ang2 was upregulated in the ischemic adductor muscle suggesting that it plays a role in recovery during hindlimb ischemia. In addition, we found that inhibiting Ang2 decreased blood flow recovery. Ang2 inhibition resulted in decreased smooth muscle cell coverage of vessels as well as decreased macrophage infiltration. These findings suggest that Ang2 promotes blood flow recovery through the recruitment of smooth muscle cells and formation of collaterals, as well as the recruitment of macrophages that secrete important growth factors and help degrade the extracellular matrix in order for neovascularization to occur. In conclusion, this work illustrates the shear stress regulation of neovessel formation through the expression of Ang2, and the role of Ang2 in neovascularization in vivo. By understanding how angiogenic factors are regulated and what role they play in vivo, we can better understand human disease and develop important therapeutic targets.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves [174]-186). Also available on the Internet.