Epithelial ovarian cancer frequently metastasizes to the omentum, a process that requires pro-angiogenic activation of local microvascular endothelial cells (ECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete factors, other than vascular endothelial growth factor (VEGF), with possible roles in metastatic angiogenesis including the lysosomal proteases cathepsin L (CathL) and cathepsin D (CathD), and insulin-like growth factor binding protein 7 (IGFBP7). However, the mechanisms by which these factors may contribute to omental endothelial angiogenic changes are unknown. Therefore the aims of this thesis were a) to examine disease relevant human omental microvascular endothelial cell (HOMEC) proliferation, migration and angiogenesis tube-formation induced by CathL, CathD and IGFBP7; b) to investigate whether CathL and CathD act via a proteolytic or non-proteolytic mechanism; c) to identify activated downstream intracellular signalling cascades in HOMECs and their activation in proliferation and migration; and finally d) to identify activated cell surface receptors by these factors. CathL, CathD and IGFBP7 significantly induced proliferation and migration in HOMECs, with CathL and CathD acting in a non-proteolytic manner. Proteome-profiler and ELISA data identified increased phosphorylation of the ERK1/2 and AKT (protein kinase B) pathways in HOMECs in response to these factors. CathL induced HOMEC proliferation and migration via the ERK1/2 pathway, whereas, although CathD-induced proliferation was mediated by activation of ERK1/2, its migratory effect was dependent on both ERK1/2 and AKT pathways. Interestingly, CathL induced secretion of galectin-1 (Gal1) from HOMECs which in turn significantly induced HOMEC proliferation via ERK1/2. However, none of the ERK1/2 or AKT pathways was observed to be active in Gal1-induced HOMEC migration. Interestingly, Gal1-induced proliferation and migration were significantly inhibited by L-glucose, suggesting a role for a receptor with extracellular sugar moieties. IGFBP7-induced migration was shown to be mediated via activation of the ERK1/2 pathway only. CathL, Gal1 and IGFBP7 significantly induced angiogenesis tube-formation in HOMECs which was not observed in CathD-treated cells. Receptor tyrosine kinase array revealed activation of Tie-1 and VEGF receptor type 2 (VEGFR2) in CathL and IGFBP7-treated HOMECs respectively. In conclusion, all CathL, CathD, Gal1 and IGFBP7 have the potential to act as proangiogenic factors in the metastasis of ovarian cancer to the omentum. These in vitro data suggest all four factors activate intracellular pathways which are involved in well-known angiogenesis models.

Orientador: Willian Fernando Zambuzzi / Resumo: As células endoteliais (ECs) e células musculares lisas (AoSMCs) são os principais componentes celulares do endotélio. As interações entre estes tipos celulares desempenham funções na homeostase e na estrutura vascular. Como uma interface entre o sangue e a parede do vaso, as ECs ocupam um local único diretamente exposto ao shear stress (SS), a força mecânica de atrito lateral produzido pelo fluxo de sangue na membrana apical da célula endotelial, que pode influenciar o comportamento de ambas ECs e AoSMCs. Geralmente, AoSMCs não sofrem diretamente às forcas de cisalhamento, no entanto, estas são diretamente expostas ao fluxo sanguíneo quando ocorre alguma injúria vascular, como por exemplo em algumas lesões ateroscleróticas ou por técnicas invasivas, como a angioplastia. As forças hemodinâmicas influenciam as propriedades funcionais do endotélio, porém estas não são profundamente compreendidas quanto aos mecanismos bioquímicos de respostas de células endoteliais e de musculatura lisa. Assim, a proposta desta dissertação foi estabelecer um modelo de cultivo in vitro que mimetize as forças tensionais de cisalhamento (shear stress), buscando compreender mecanismos celulares, bioquímicos e epigenéticos. Cultura de células primárias endoteliais e de musculatura lisa humanas foram obtidas da empresa LONZA e mantidas conforme recomendações do fabricante. Estas células foram mantidas rotineiramente em condições convencionais em incubadora de CO2. Para mimetizar o fluxo sanguíneo, esta... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

As doenças cardiovasculares ainda são as principais causas de mortalidade e morbidade em todo o mundo. E a aterosclerose é uma das principais precursoras de vários desfechos clínicos como isquemias e infarto do miocárdio. As placas ateroscleróticas se desenvolvem preferencialmente em regiões de bifurcação ou curvatura dos vasos, onde o shear stress (SS) encontra-se diminuído ou perturbado. A expressão de proteínas pró-aterogênicas em regiões de baixo SS e ateroprotetoras em regiões de SS alto foram relatadas na literatura, porém o mecanismo completo carece de elucidação. Este trabalho teve por objetivo integrar proteômica e metabolômica para um melhor entendimento das alterações moleculares que acontecem nas células endoteliais em situações de alto e baixo SS, que podem resultar no desenvolvimento de lesões e placas ateroscleróticas. Para esta finalidade, células endoteliais foram submetidas a alto e baixo SS em sistema cone plate, seguido de análise proteômica e metabolômica por espectrometria de massas. Nossos dados demonstraram que o metabolismo de lipídio e metabolismo de modificações pós-traducionais de proteínas (N-glicosilações) estavam diminuídos em baixo SS. Em relação ao metabolismo de lipídio, foi identificada diminuição na concentração de ácidos graxos e na expressão de enzimas e proteínas transportadoras de lipídios em células sob baixo SS. O receptor de LDL, proteína importante para a homeostase do colesterol, foi identificado em menor concentração na membrana, bem como com alteração no seu perfil de glicosilação em células após baixo SS. As células submetidas a baixo SS e, portanto, aquelas com perfil pró-aterogênico, quando tratadas com estatina para o aumento da expressão de LDLR, aproximaram seu fenótipo ao de células submetidas a alto SS, adquirindo parte de um fenótipo ateroprotetor, com recuperação dos níveis de aminoácidos, lipídios, açúcares e ácidos carboxílicos. Os dados deste trabalho sugerem que o metabolismo de lipídios é um processo importante na manutenção do perfil ateroprotetor de células submetidas a alto SS. Além disso, as evidências demonstraram que estatinas apresentam uma atividade protetora, não apenas sistêmica, com diminuição do LDL circulante, mas também no microambiente vascular, contribuindo para o bom funcionamento das células endoteliais / Cardiovascular diseases are the main cause of the mortality and morbidity worldwide. Atherosclerotic plaque development is closely associated to the hemodynamic forces applied to endothelial cells (EC). Among these, shear stress (SS) plays a key role in disease development since changes in flow intensity and direction could stimulate an atheroprone or atheroprotective phenotype. EC under low and/or oscillatory SS (LSS) have upregulation of inflammatory proteins, adhesion and cellular permeability molecules. On the contrary, cells under high/laminar SS (HSS) increase their expression of protective and anti-inflammatory factors. The mechanism behind the SS regulating an atheroprotective phenotype is not completely elucidated. Here we used proteomics and metabolomics to better understand the changes suffered by endothelial cells under LSS and HSS that promote the atheroprone and atheroprotective profile and how these modifications can be connected to atherosclerosis development. Our data showed that lipid metabolism and post translational modification protein metabolism were downregulated in cells under LSS. About lipid metabolism, we found the LDLR, one important protein in cholesterol homeostasis, showed significant alterations both at the quantitative expression level, as well as regarding post-translational modifications. Under LSS, LDLR was seem at lower concentrations and with a different glycosylation profile. Finally, modulating LDLR with atorvastatin led to the recapitulation of an HSS metabolic phenotype in EC under LSS. The phenotype was recovery based on increasing of amino acids, lipids, sugars and carboxylic acids. Altogether, our data suggest lipid metabolism is important in atheroprotective phenotype of endothelial cells under HSS. Statins showed benefits not only systemic, decreasing cholesterol level in blood, but also in vascular environment, contributing for protector phenotype of endothelial cells

Os riscos provocados pela Terapia de Reposição Hormonal, levaram à busca de novas terapias, como os fitoestrógenos. São substâncias com ação estrogênica e propriedades que podem retardar a formação de placas ateroscleróticas. Isoflavonas são os fitoestrógenos mais estudados e são encontradas na soja, no red clover e em outras plantas. Avaliamos os efeitos dos fitoestrógenos extraídos da soja Glycine max: genisteína, formononetina, biocanina A e daidzeína; a mistura entre eles (Mix1); o extrato padronizado de red clover (Menoflavon 40mg) e uma segunda mistura com os fitoestrógenos extraídos da Glycine max nas concentrações encontradas no Menoflavon (Mix2), na expressão de moléculas de adesão de leucócitos, VCAM-1, ICAM-1 e E-selectina, em cultura de células endoteliais de cordão umbilical humano (HUVEC), assim como na linhagem modificada de célula endotelial, ECV304, estimuladas com LPS. Resultados: foram padronizados os tempos e concentrações de exposição ao LPS no cultivo de HUVEC de 1ug durante 12 horas de exposição para as três moléculas de adesão; e no cultivo de ECV304 para a expressão de VCAM-1, de 500ng durante 12 horas, para ICAM-1 de 1ug durante 18 horas, para E-selectina 100ng durante 18 horas na superfície celular e 200ng durante 24 horas no sobrenadante de culturas de ECV304, permitindo que este tipo celular seja utilizado como modelo de inflamação. Os fitoestrógenos reduziram VCAM-1, ICAM-1 e E-selectina na superfície celular assim como as formas solúveis dessas moléculas, tanto em ECV304 como em HUVEC, sendo efetivos como agentes preventivos e também para tratamento da aterosclerose. A mistura entre os fitoestrógenos não apresentou maior eficiência na redução das moléculas de adesão na superfície celular, mas apresentou diferenças significativas na produção das formas solúveis. Tanto em ECV304, quanto em HUVEC os fitoestrógenos extraídos do red clover e os extraídos da soja Glycine max reduziram as moléculas de adesão na superfície celular e no sobrenadante, sendo que o Menoflavon, apresentou maior efetividade na redução das moléculas de adesão que os fitoestrógenos extraídos da soja Glycine max, em HUVEC. Ocorreram interações entre os fitoestrógenos e o 17 estradiol, tanto em ECV304 quanto em HUVEC, principalmente quando este se encontrava em baixas concentrações, sugerindo proteção para mulheres na menopausa. Esses efeitos dos fitoestrógenos ocorreram via receptor de estrógeno, como demonstrado pela inibição de suas ações por ICI. Conclusão: tanto os fitoestrógenos extraídos da soja Glycine max quanto os extraídos do red clover apresentaram efeitos anti-aterogênicos, podendo atuar como cardioprotetores para mulheres pós-menopausa. / The risks of hormone replacement therapy have led to a search for new alternatives such as the use of phytoestrogens, plant compounds with estrogen-like biological activity. Isoflavones are the phytoestrogens most extensively studied and can be found in soy, red clover and other plants. Due this estrogen-like activity phytoestrogens can have some effect on atherosclerosis. We evaluated the effects of the phytoestrogens extracts from Glycine max soy: genistein, formononetin, biocanin A and daidzein; a Mix between them (Mix1); a standardized red clover extracts (Menoflavon 40mg) and a second Mix using phytoestrogens from Glycine max with same Menoflavon concentrations (Mix2) on adhesion molecules expression, VCAM-1, ICAM-1 and E-selectin by endothelial cell HUVEC, and by endothelial cell line ECV304, stimulated with LPS. Results: were standardized time and concentration to LPS exposure, being 1ug during 12 hours for the three adhesion molecules expression on HUVEC, and 500ng during 12 hours for VCAM-1 expression, 1ug during 18 hours for ICAM-1 expression and 100ng during 18 hours for E-selectin expression on cell surface as well as 200ng during 24 hours to E-selectin increase on culture supernadant, on ECV 304 cell line. The phytoestrogens decreased VCAM-1, ICAM-1 and E-selectin levels on cell surface and on culture supernadant in HUVEC and ECV304, being useful as preventive agents as well as treatment agents. Mix1 were not most effective than isolated phytoestrogens on cell surface, but presented decreased results on soluble forms. Menoflavon presented more effectiveness than Glycine max on HUVEC. Phytoestrogens interacted with 17 oestradiol mainly, in low concentrations (10pg), showing protection for post menopausal women. These phytoestrogens effects happened by oestrogen receptor activation, this was demonstrated through phytoestrogens inhibition by ICI. Conclusions: the phytoestrogens from Glycine max as well as phytoestrogens from red clover presented antiatherogenic effects, mainly when 17 estradiol is low, being usefull for postmenopausal women.

Diabetic foot ulceration (DFU) is a known major complication of diabetes mellitus which contributes to lower extremities amputation. This study aimed to investigate the use of interventional devices either as a preventative or therapeutic strategy to improve clinical management of this pathology, as well as investigating the impaired function of endothelial progenitor cells in the diabetic condition. The first element targeted plantar callus formation among diabetic neuropathic (NRP) patients through the use of a SurroSenseRxTM biofeedback device. Reducing foot pressure with improved walking strategy in the 6 months study in diabetic neuropathy patients (n=20) appeared to minimise the size of non-ulcerative plantar callus (p < 0.05), potentially reducing future ulcer recurrence. The 2nd study focused on the use of a GekoTM electrical stimulation device to enhance DFU healing in 24 patients. Wounds were characterised as being neuroischaemic (NRI) or neuropathic (NRP) based on standard parameters adopted in the Manchester diabetes clinic. The device was worn by 11 intervention subjects and compared to 13 controls without any electrical stimulus. Results suggested healing and wound closure have potentially increased in participants with electrical stimulation. In addition, Neuropathy Disability Score (NDS) was improved among intervention patients compared to control (p < 0.0001). The 3rd, in vitro and mechanistic study focuses on the outgrowth of endothelial cells (OECs), abnormal angiogenic responses and inflammatory microenvironment which could contribute to impaired wound healing in diabetic patients. OECs were isolated from diabetic patients and healthy controls (HCs), characterised by immunohistochemistry and Polymerase Chain Reaction (PCR). The functions of the three OEC groups from NRI, NRP diabetic patients and healthy controls respectively were compared using in vitro proliferation, transwell migration and wound healing scratch assays, together with matrigel tube formation assays. Scratch assays showed 100% closure in HCs over 24 hours, while 86.6% closure was apparent in NRI vs 38.1% in NRP. Seahorse mitochondrial stress test was conducted and demonstrated mitochondrial dysfunction in NRP vs NRI vs HCs (p < 0.05). Western blot analysis showed a lack of ERK phosphorylation by NRP OECs and an up-regulation of plasma inflammatory cytokines (TNFa and IL-6) in diabetic samples vs HC (p < 0.0001), while the angiogenic factors ang-2, FGF-2, VEGF-D, HGF and IL-8, and nitric oxide bioavailability were all significantly reduced in diabetic samples vs HC (p < 0.05). The functional defects of the diabetic OECs were partially restored through glycomimetic (synthesis compounds for endothelial damage protection) treatment (p < 0.05). In summary, this study has highlighted areas worthy of future development both in terms of preventative and therapeutic strategies. With improvements in digital technology and the need to empower patients to take responsibility of their health and well-being as well as greater understanding of the cellular and molecular biological repair processes that may be exploited, there may be potentials to reduce the risk of future ulceration among patients using these novel approaches in the future.

O melanoma é uma neoplasia de pele invasivo, com maior taxa de morte, sem tratamento efetivo. Nanocápsulas poliméricas de núcleo lipídico (LNC) tem sido empregadas com sucesso como carreadores de fármacos hidrofóbicos. Como o eugenol é um composto hidrofóbico com atividades antiproliferativas e pró-apoptóticas em células cancerosas, visamos avaliar os efeitos dos tratamentos com acetileugenol (AC), LNC ou LNC contendo acetileugenol (LNC-AC) em modelo de melanoma in vivo em camundongos C57B6, e a citotoxicidade dos mesmos em células endoteliais (HUVEC) e de melanoma (SK-Mel-28) in vitro. Os resultados obtidos mostraram que: 1) tratamentos i.p. com as LNC ou com LNC-AC (50 mg/kg, 3-10 dia de indução do tumor) induziram toxicidade sistêmica e, somente o tratamento com LNC inibiu o desenvolvimento do melanoma. O tratamento com LNC, mas não com a mistura de triglicerídeos de cadeia média, por via oral, inibiu o desenvolvimento tumoral, sem toxicidade. Adicionalmente, os tratamentos com AC, LNC ou LNC-AC não foram eficazes quando administrados em fase tardia de evolução tumoral (50 mg/kg, 7-17 dia de indução do tumor, via oral); 2) os tratamentos agudos com AC, LNC ou LNC-AC (20 mg/kg, 200 &#181;L, e.v.) não alteraram o número de leucócitos circulantes, mas os tratamentos com LNC ou com LNC-AC reduziram o comportamento de rolling dos leucócitos em vênulas póscapilares do músculo cremaster e causaram hemólise, sendo que este último efeito também foi observado após tratamento in vitro em hemácias murinas; 3) Os estudos in vitro mostraram que as LNC e LNC-AC foram captadas pelas células HUVEC e SK-Mel-28 após 1 hora de incubação; que a incubação com LNC-AC induziu apoptose tardia e necrose com maior eficácia em SK-Mel-28 do que em HUVEC; que as incubações com LNC ou LNC-AC exerceram efeitos antiproliferativos, induzindo parada na fase G2/M do ciclo celular das duas linhagens de células avaliadas; que somente a incubação com AC ou LNC-AC inibiu a adesão ao Matrigel® com maior eficácia na linhagem SK-Mel-28 do que HUVEC; que somente a incubação com as LNC reduziram a expressão de VCAM-1 em HUVEC e que as incubações com LNC ou LNC-AC reduziram a expressão de &#946;3 integrina em SK-Mel- 28; que nenhum dos tratamentos alterou a migração celular das HUVEC ou SK-Mel- 28; que somente a incubação com LNC-AC reduziu os níveis de espécies reativas de oxigênio em HUVEC e SK-Mel-28; que a incubação com LNC ou LNC-AC aumentou a produção de óxido nítrico (NO) pelas duas linhagens de células avaliadas; que o tratamento com L-NAME reverteu os níveis de NO e a inibição sobre a proliferação celular induzida pela incubação com LNC ou LNC-AC e; que o tratamento de células de melanoma murino com LNC ou LNC-AC parece alterar a polarizar os neutrófilos para o fenótipo N1. Associados, os resultados obtidos mostram o tratamento oral com LNC inibe o crescimento do melanoma sem induzir efeitos tóxicos, e que este efeito benéfico pode ser dependente, pelo menos em parte, da nanoencapsulação dos triglicerídios de cadeia média e da supraestrutura da formulação, com toxicidade direta sobre as células de melanoma e possível modulação do microambiente tumoral. / Melanoma is the most invasive skin cancer, with high rates of death without effective treatment. Polymeric lipid-core nanocapsules (LNC) has been successfully used as carriers of hydrophobic drugs. As eugenol is an hydrophobic compound with antiproliferative and pro-apoptotic activity in cancer cells, here we aimed to evaluate the effects of treatments with acetyleugenol (AC), LNC or LNC containing acetyleugenol (LNC-AC) in an in vivo melanoma model in C57BL6 mice and the cytotoxicity of the treatments in vitro, using endothelial (HUVEC) and melanoma (SK-Mel- 28) cells. The results obtained showed that: 1) i.p. treatments with LNC or LNCAC (50 mg/kg, 3-10 days of tumor injection) induced systemic toxicity and, only the treatment with LNC inhibited the melanoma development. Treatment with LNC, but not with mix of triglycerides of medium chain, by oral route, inhibited the tumor development, without toxicity. In addition, the treatments with AC, LNC or LNC-AC were not effective when administered in the late stage of tumor evolution (50 mg/kg, 10-20 days of tumor induction, oral route); 2) the acute treatments with AC, LNC or LNC-AC (20 mg/kg, 200 &#181;L, intravenous route) did not altered the number of circulating leukocytes, but the treatments with LNC or LNC-AC reduced the rolling behavior of leukocytes in postcapillary venules of the cremaster muscle and induced hemolysis. The latter effect was also observed after in vitro treatment using murine erythrocytes; 3) In vitro studies showed that the LNC and LNC-AC suffered uptake by HUVEC and SK-Mel-28 cells after 1 hour of incubation; that the incubation with LNC-AC induced late apoptosis and necrosis more effectively in SK-Mel-28 than in HUVEC cells; that the incubation with LNC or LNC-AC presented antiproliferative effects, by inducing G2M arrest in cell cycle in both cells lines evaluated; that only the incubation with AC or LNC-AC inhibited the adhesion in Matrigel® with more efficaccy in SK-Mel-28 than in HUVEC cells; that only incubtion with LNC reduced the VCAM-1 expression in HUVEC and the incubation with LNC or LNC-AC reduced the &#946;3 integrin expression in SK-Mel-28 cells; that any treatment affected the HUVEC or SK-Mel- 28 migration; that only the incubation with LNC-AC reduced the levels of reactive species of oxygen in HUVEC and SK-Mel-28 cells; that the incubation with LNC or LNC-AC increased the nitric oxide (NO) production by both cell lines used; that the treatment with L-NAME reversed the NO levels and the inhibition on cell proliferation induced by incubation with LNC or LNC-AC and; that the in vitro treatment of murine with LNC or LNC-AC altered the neutrophil polarization to N1 phenotype. Together, results obtained show that the oral treatment with LNC inhibit the melanoma growth without any toxic effect, and that the beneficial effect could be dependent, at least in part, of nanoencapsulation of medium chain triglycerides and the supraestrucuture of the formulation, with direct toxicity on melanoma cells and possible modulation of tumor microenvironment.

Cerebral small vessel disease (SVD) is a vascular neurodegenerative disease which is the leading cause of vascular dementia and causes 20% of strokes. 20-30% of those over 80 show signs of the disease as white matter hyperintensities on MRI scans, doubling their risk of stroke and trebling their risk of dementia. Sporadic SVD is thought to be caused by hypertension but 30% of sufferers are normotensive and an alternative hypothesis implicates loss of integrity of the blood brain barrier (BBB). To investigate this, I studied brains from normotensive people with early stage SVD and found reduced capillary endothelial claudin-5 (a BBB tight junction protein), more oligodendrocyte precursor cells (OPCs; the precursors to myelinating oligodendrocytes), and more microglia/macrophages compared to controls. Furthermore, in a relevant rat model of spontaneous SVD, the Stroke Prone Spontaneously Hypertensive Rat (SHRSP; disease model; DM) I found that reduced endothelial claudin-5 was the earliest change, appearing at 3 weeks of age, followed by OPC proliferation, appearing at 4 weeks, and then increased number of microglia/macrophages, appearing at 5 weeks. Importantly, all these changes occurred at a young age (< 5 weeks), before any measurable hypertension. These changes were confirmed in an ex vivo slice culture model (i.e. removing blood flow), ruling out direct damage by leakage of blood components through an impaired BBB and suggesting an inherent endothelial cell dysfunction as the primary cause, with secondary BBB defects. This hypothesis of endothelial dysfunction is supported by increased endothelial cell proliferation in both human SVD tissue and the DM rats, and lower levels of endothelial nitric oxide synthase (eNOS) in brains of DM rats. To study this further I isolated primary brain microvascular endothelial cells (BMECs) from DM and control rats and found that those from DM rats formed less mature tight junctions (less membranous claudin-5) than control BMECs. I also found that conditioned media (CM) from DM BMECs causes OPCs in culture to proliferate more and mature less. This indicates that the endothelial dysfunction is inherent to the endothelial cells, rather than induced by other cell types, and through secreted factors causes OPC changes mirroring what is seen in vivo. Using an antibody array, I identified HSP90α as a candidate secreted factor and showed that it is necessary (by blocking the protein in CM) and sufficient (by adding recombinant HSP90α) to induce the maturation phenotype in OPCs, but not the proliferation phenotype. The idea that endothelial dysfunction causes SVD begs the question of what causes endothelial dysfunction, especially in our inbred DM rat strain. To establish this, I reanalysed sequencing data of the DM and control rats from a previously published study, searching for mutations which lead to truncated proteins in genes expressed in brain endothelial cells. We confirmed the candidate gene Atp11b, a phospholipid flippase, was mutated as predicted. I found that knocking down Atp11b using siRNA in a control endothelial cell line caused endothelial dysfunction and a loss of tight junction maturity, and that CM from these cells causes OPCs to proliferate more and mature less, mirroring what we see in primary DM BMECs and suggesting that Atp11b has a key function in promoting normal endothelial function. Furthermore, I showed that knocking down Atp11b causes cells to secrete increased levels of HSP90α. I propose a mechanism whereby ATP11B regulates the retention of HSP90α within endothelial cells, which in turns regulates eNOS levels and activity, as has been shown previously. In summary, this work shows that there are many pre-symptomatic changes which occur in the brain in the development of SVD in DM rats, and that these are ultimately caused by endothelial dysfunction. As these changes are similar to those found in spontaneous human SVD, I propose that endothelial dysfunction is a key mechanism of human SVD, which may in the future lead to new therapies.

The hamster estrous cycle lasts four days and is considered to be a physiological model for angiogenesis. Angiogenesis is the formation of new capillaries from preexisting vessels, and it occurs extensively during corpus luteum formation in the estrous cycle. Von Willebrand Factor (vWF) is a glycoprotein that is secreted uniquely in endothelial cells and megakaryocytes. It is frequently used as an endothelial cell marker and it is able to detect vessels within tissues when it is used in immunohistochemical staining techniques. This study explores von Willebrand Factor expression within Golden Hamster ovarian tissue. In particular, this study uses cage control and antiorthostatic cage suspension tissue. Antiorthostatic cage suspension is a model developed to mimic and study the physiological effects caused by microgravity, such as that experienced in space flight. It is hypothesized that simulated microgravity caused by antiorthostatic cage suspension would result in lower levels of vasculature and expression of vWF within ovarian tissue. Due to financial considerations, conclusive data was not obtained due to a lack of statistics. However, our study indicates that vasculature and vWF expression may be increased in antiorthostatic cage suspension tissue.

Previously, we cloned Heat shock protein A12B (HspA12B), the newest member of a recently defined subfamily of proteins distantly related to the Hsp70 family that are enriched in atherosclerotic lesions. We have found that HspA12B is predominantly expressed in vascular endothelium, and that it is involved in angiogenesis which we probed by in vitro angiogenesis assays (Matrigel), migration assays and Directed In Vivo Angiogenesis Assay (DIVAA). Hsp70s are molecular chaperones that are inducible by stress and have been found to be anti-apoptotic (Li et al. 2000; Nylandsted et al. 2000; Garrido et al. 2001). Because of its homology to Hsp70, we propose that it is the first endothelial-specific chaperone that is required for angiogenesis and interacts with known angiogenesis regulators. To begin to understand the molecular mechanisms underlying the role of HspA12B in angiogenesis, we turned our attention to identifying proteins that are involved in angiogenesis and also interact with HspA12B. Through the use of a yeast two-hybrid (Y2H) system HspA12B was found to interact with a known angiogenesis regulator, A Kinase Anchoring Protein 12 (AKAP12). This interaction was confirmed by co-immunoprecipitation and by colocalization. In primary human umbilical vein endothelial cells (HUVECs), shRNA mediated HspA12B knockdown increased AKAP12 levels and decreased VEGF by more than 75%, whereas HspA12B over-expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-Amino Acid (32-AA) domain in AKAP12 that mediates interaction with HspA12B. Over-expression of this 32-AA domain in HUVECs disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression suggesting the importance of the HspA12B-AKAP12 interaction in regulating VEGF. This is the first evidence that HspA12B promotes angiogenesis resulting in up-regulation of VEGF by suppressing AKAP12. Consistent with the proposed role in angiogenesis, HspA12B was also found to be increased in endothelial cells (ECs) by angiogenic stresses including hypoxia and shearing stress while knockdown of HspA12B abolished hypoxia-induced tubule formation. This work provides new insight into the mechanisms controlling angiogenesis by providing the first example of an EC-specific molecular chaperone that acts as a regulator of angiogenesis and lays the foundation for future studies of HspA12B-derived therapeutics for angiogenesis related diseases.

Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.