In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

Die Bedeutung der angiogenen Faktoren „soluble fms-like tyrosine kinase-1“ (sFlt-1) und „placental growth factor“ (PIGF) in der Pathogenese der Präeklampsie (PE) ist in den letzten Jahren hinreichend belegt worden. Bei Patienten mit manifester PE lassen sich dramatisch hohe sFlt-1- bzw. sehr niedrige PlGF-Werte nachweisen. Mittlerweile konnte in Studien die sFlt-1/PlGF-Ratio als sensitiver Parameter ermittelt werden. Ziel der vorliegenden Arbeit ist es, die Zusammenhänge zwischen klinischen Parametern der manifesten Präeklampsie und der sFlt-1/PlGF-Ratio zu spezifizieren. Dabei soll geprüft werden, ob bei manifester PE die Höhe der sFlt-1/PlGF-Ratio vor Entbindung klinische Bedeutung hat und mit Parametern des Schwangerschaftsausgangs in Beziehung steht.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

A brief introduction to cancer biology and treatment is presented with a focus on current clinical advances in the delivery of chemotherapy and antiangiogenic therapies. Mathematical oncology is then surveyed with summaries of various models of tumor growth, tumor angiogenesis and other relevant biological entities such as angiogenic growth factors. Both strictly time-dependent ordinary differential equation (ODE)-based and spatial partial differential equation (PDE)-based models are considered. These biological models are first developed into an ODE model where various treatment options can be compared including different combinations of drugs and dosage schedules. This model gives way to a PDE model that includes the spatially heterogeneous blood vessel distribution found in tumors, as well as angiogenic growth factor imbalances. This model is similarly analyzed and implications are summarized. Finally, including the effects of interstitial fluid pressure into an angiogenic activity model is performed. This model displays the importance of factor convection on the angiogenic behaviour of tumours.

The tumor microenvironment consists of multiple cells types, including endothelial cells that line the tumor vasculature. Tumor vasculature is often abnormal and results in development of tissue ischemia, another contributing factor to the tumor microenvironment. Previous studies have demonstrated that ischemia influences epigenetic programming, but the mechanisms remained unclear and required further investigation. First, we profiled DNA methyltransferase (DNMT) expression and activity in human colorectal cancer cells (HCT116) under hypoxia or hypoglycaemia (mimicking ischemia). We found that DNMT1 and DNMT3b were significantly downregulated by hypoxia and hypoglycaemia, and DNMT3a was downregulated by hypoglycaemia. However, DNMT1 downregulation was p53-dependent. To examine if the changes in DNMT expression and activity translated to changes in DNA methylation patterns, we used bisulfite sequencing and examined the promoter region of p16. Hypoglycaemia significantly demethylated this region in both p53 wild-type and p53-null cells. Next, we used a genome-wide approach to discover what additional genes are hypomethylated by ischemia. Methylated DNA was immunoprecipitated and analysed with an Affymetrix promoter array, in parallel with an expression array. Ingenuity pathway analysis software revealed that a significant proportion of genes which were hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. We believe that hypoxia and hypoglycaemia may be driving changes in DNA methylation through dysregulation of DNMTs, resulting in cells acquiring a more mobile phenotype in ischemic regions. DNMT and histone deacetylase inhibitors are commonly used in research and some cancer therapies. Modifying epigenetic patterning with these inhibitors has been widely studied in cancer cells, but only briefly explored in the tumor’s vascular endothelium. We profiled the effect of these inhibitors on endothelial cell (EC) behaviour, and tested if combining them with a targeted anti-angiogenic therapy would augment the inhibition of angiogenesis. When the DNMT inhibitor 5-aza-2’-deoxycytidine was combined with sunitinib, inhibition of EC proliferation was enhanced compared to treatment with sunitinib alone. EC migration was also inhibited by the combination of these two inhibitors, but not in an additive manner. These studies have improved our understanding of how altering epigenetic patterning with ischemia and therapeutic inhibitors can influence colorectal cancer and endothelial cell behaviour. / Canadian Cancer Society Research Institute. The Cancer Research Society.

The expanding field of tissue engineering provides a new approach to regenerative medicine for common ailments such as cardiovascular disease and type-I diabetes. Biomaterials can be administered as a delivery vehicle to introduce therapeutic cells to sites of damaged or diseased tissue. A specific class of biomaterials, termed hydrogels, is suitable for this application as they can provide a biocompatible, biodegradable scaffold that mimics the physical properties of the native soft tissue. Injectable hydrogels are increasingly being developed for biomedical applications due to their ability to be delivered in a minimally invasive manner. One potential use for such materials is in the delivery of therapeutics such as cells or growth factor-releasing particles. In this study, the first aim was to determine the interactive effects between collagen-based hydrogels and additives (cells and microspheres) for cardiac regeneration. The results demonstrated that the addition of either cells or microspheres to a collagen-based hydrogel decreased its gelation time and increased its viscosity. Increased cross-linker concentrations resulted in lower cell viability. However, this cell loss could be minimized by delivering cells with the cross-linker neutralizing agent, glycine. As a potential application of these materials, the second aim of this study was to develop a hydrogel for use as an ectopic islet transplant site. Specifically, collagen-chitosan hydrogels were synthesized and characterized, with and without laminin, and tested for their ability to support angiogenic and islet cell survival and function. Matrices synthesized with lower chitosan content (20:1 collagen:chitosan) displayed greater cell compatibility for both angiogenic cells and for islets and weaker mechanical properties, while matrices with higher chitosan content (10:1 collagen:chitosan) had the opposite effect. Laminin did not affect the physical properties of the matrices, but did improve angiogenic cell and islet survival and function. Overall the proposed collagen-based hydrogels can be tailored to meet the physical property requirements for cardiac and islet tissue engineering applications and demonstrated promising cell support capabilities.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references (p. 99-116).

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-08T13:25:55Z No. of bitstreams: 1 arquivototal.pdf: 2508821 bytes, checksum: 881e48cd3cb91d680602af2fa3e62a64 (MD5) / Made available in DSpace on 2017-09-08T13:25:55Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 2508821 bytes, checksum: 881e48cd3cb91d680602af2fa3e62a64 (MD5) Previous issue date: 2015-02-27 / Cancer is characterized by uncontrolled proliferation of abnormal cells. The high mortality rate associated with the disease, coupled to severe side effects caused by conventional treatments and the increasing development of resistance, has conducted to the demand for effective therapies with less toxicity. In this context, it is of great relevance the synthesis of synthetic products with structures based on natural products, which continue to be the main source of new medicines. In order to increase the effect and minimize the toxicity of piperine, an amide alkaloid with antitumor activity, it was obtained novel synthetic analogues, among them the 2-oxo-2-(4-ethylamine)-piperinoato acetate (HE-03). This study aimed to investigate the antitumor activity and toxicity of HE-03. The data demonstrate that HE-03 induced a small percentage of hemolysis to the highest concentration tested (1250 mg/mL), which suggests low toxicity to erythrocytes. In the preclinical acute toxicity study, the treatment with doses of 300 mg/kg and 2000 mg/kg of HE-03 in mice caused no deaths, and the changes observed in the CNS and ASN level were transient (30 min.). The LD50 value was estimated to be about 5000 mg/kg. HE-03 (6.25, 12.5 and 25 mg/kg) showed significant antitumor activity in vivo in ascites Ehrlich carcinoma model, especially considering the viability parameters and total cell. Its effect in doses of 12.5 and 25 mg/kg was similar to the one observed for the anticancer drug 5-fluorouracil (25 mg/kg). Cell cycle profile analysis showed that treatment with HE-03 (12.5 mg/kg) induced the appearance of sub-G1 peak, which was accompanied by a reduction in the number of cells in G0/G1 and S phases, suggesting induction of cell death by apoptosis. The treatment with HE-03 (12.5 mg/kg) significantly reduced peritumoral microvessels, which suggests anti-angiogenic activity. The toxicological analyzes indicate that the nine days of treatment with HE-03 at all doses, induced a significant increase in serum aspartate aminotransferase (AST) and urea. However, histopathology of the liver and kidneys showed that HE-03 does not induce changes of clinical importance. Leukopenia, accompanied by neutropenia, observed in animals treated with 25 mg/kg of HE-03 is one of the major side effects associated with cancer therapy. Futhermore, HE-03 did not induce an increase in the number of micronucleated erythrocytes in the micronucleus assay in peripheral blood, which indicates lack of genotoxicity in the evaluated conditions. Therefore, it is possible to infer that the synthetic analogue of studied piperine has potent antitumor activity in vivo, with mechanism that possibly involves the induction of apoptosis and antiangiogenic effects, as well as low toxicity. / O câncer caracteriza-se pela proliferação descontrolada de células. A elevada taxa de mortalidade associada à doença, aliada aos graves efeitos indesejáveis causados pelos tratamentos convencionais e ao crescente desenvolvimento de resistência, tem impulsionado a procura por terapias mais eficazes com menor toxicidade. Nesse contexto, é de grande relevância a síntese de produtos sintéticos com estruturas baseadas em produtos naturais, que continuam a ser a principal fonte de novos medicamentos. Com o intuito de potencializar o efeito e minimizar a toxicidade da piperina, um alcaloide amida com atividade antitumoral, foram obtidos análogos sintéticos inéditos, dentre estes o 2-oxo-2-(4-etilamina)-piperinoato de etila (HE-03). Esse trabalho objetivou investigar a atividade antitumoral, bem como a toxicidade do HE-03. Os dados demonstram que HE-03 induziu pequeno percentual de hemólise até a maior concentração testada (1250 μg/mL), o que sugere baixa toxicidade nos eritrócitos. No ensaio de toxicidade pré-clínica aguda, o tratamento com as doses de 300 mg/kg e 2000 mg/kg do HE-03 não provocou mortes nos camundongos, e as alterações observadas em nível de SNC e SNA foram e de curta duração (30 min.) e sem significância no tempo observado. O valor da DL50 foi estimado em torno de 5000 mg/kg. HE-03 (6,25; 12,5 e 25 mg/kg) mostrou significante atividade antitumoral in vivo em modelo de carcinoma ascítico de Ehrlich, considerando especialmente os parâmetros viabilidade e total celular. Seu efeito nas doses de 12,5 e 25 mg/kg foi semelhante ao observado para o antineoplásico 5-fluorouracil (25 mg/kg). Na análise do perfil do ciclo celular, foi observado que o tratamento com HE-03 (12,5 mg/kg) induziu o aparecimento do pico sub-G1, que foi acompanhado por uma redução na quantidade de células nas fases G0/G1 e S, o que sugere indução de morte celular por apoptose. O tratamento com HE-03 (12,5 mg/kg) reduziu significativamente a microdensidade vascular peritumoral, o que sugere atividade antiangiogênica. As análises toxicológicas indicam que o tratamento de nove dias com HE-03, em todas as doses, induziu aumento significativo de aspartato aminotransferase (AST) e de ureia. Todavia, a histopatologia do fígado e dos rins mostrou que HE-03 não induz alterações de importância clínica. A leucopenia, acompanhada de neutropenia, observadas nos animais tratados com 25 mg/kg de HE-03 é um dos principais efeitos indesejáveis associados a terapia do câncer. Ainda, HE-03 não induziu aumento no número de eritrócitos micronucleados, no ensaio do micronúcleo em sangue periférico, o que indica ausência de genotoxicidade, nas condições avaliadas. Portanto, é possível inferir que o análogo sintético da piperina estudado apresenta potente atividade antitumoral in vivo, com mecanismo que envolve possivelmente a indução de apoptose e efeitos antiangiogênicos, bem como, baixa toxicidade.