Sy Chun Choi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 123-160). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / Chapter 1. --- Introduction / Chapter 1.1 --- General review of the testis --- p.1 / Chapter 1.1.1 --- Structure and function of the testis --- p.1 / Chapter 1.1.2 --- Testicular vasculature --- p.2 / Chapter 1.1.3 --- Testicular angiogenesis --- p.3 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.4 / Chapter 1.2.1 --- Discovery of VEGF --- p.4 / Chapter 1.2.2 --- Organization of VEGF --- p.4 / Chapter 1.2.3 --- Properties of the VEGF isoforms --- p.5 / Chapter 1.2.4 --- VEGF receptors --- p.6 / Chapter 1.2.5 --- Functions of VEGF --- p.8 / Chapter 1.3 --- VEGF in the testis --- p.10 / Chapter 1.3.1 --- Localization of VEGF and VEGF receptors in the testis --- p.10 / Chapter 1.3.2 --- Postulated functions of VEGF in the testis --- p.11 / Chapter 1.4 --- Regulation of VEGF --- p.11 / Chapter 1.4.1 --- "VEGF, hypoxia and the testis" --- p.11 / Chapter 1.4.2 --- "VEGF, nitric oxide and the testis" --- p.14 / Chapter 1.4.3 --- Cadmium-induced testicular toxicity --- p.16 / Chapter 1.4.4 --- "VEGF, glucocorticoids and the testis" --- p.17 / Chapter 1.4.5 --- Hormonal regulation of VEGF expression 226}0ؤimportance of LH --- p.19 / Chapter 1.4.6 --- VEGF and Leydig cell - macrophage interaction --- p.21 / Chapter 1.5 --- Aims of the present study --- p.24 / Chapter 2. --- Materials and methods / Chapter 2.1 --- Animals --- p.26 / Chapter 2.1.1 --- Spermatic cord torsion --- p.26 / Chapter 2.1.2 --- Cadmium chloride treatment --- p.27 / Chapter 2.1.3 --- Leydig cell depletion and cadmium chloride treatment --- p.28 / Chapter 2.1.4 --- Dexamethasone pretreatment and cadmium chloride injection --- p.28 / Chapter 2.1.5 --- hCG injection --- p.29 / Chapter 2.2 --- Immunohistochemistry --- p.29 / Chapter 2.2.1 --- Perfusion fixation of the testes --- p.29 / Chapter 2.2.2 --- Processing of the testes for histological section --- p.29 / Chapter 2.2.3 --- Immunohistochemical staining for VEGF --- p.30 / Chapter 2.2.4 --- Photomicrograph --- p.32 / Chapter 2.3 --- Cell cultures --- p.32 / Chapter 2.3.1 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.32 / Chapter 2.3.2 --- Tumour cell line of mouse MLTC-1 Leydig cells --- p.33 / Chapter 2.4 --- Cell treatments --- p.33 / Chapter 2.4.1 --- Hypoxic treatment --- p.34 / Chapter 2.4.2 --- Cadmium chloride treatment --- p.36 / Chapter 2.4.3 --- hCG treatment --- p.37 / Chapter 2.4.4 --- Activators of second messenger systems --- p.37 / Chapter 2.4.5 --- Effect of pro-inflammatory cytokines and angiogenic growth factors --- p.38 / Chapter 2.5 --- Preparation of cDNA probes --- p.39 / Chapter 2.5.1 --- VEGF cDNA probe preparation --- p.39 / Chapter 2.5.2 --- P-actin cDNA probe preparation --- p.42 / Chapter 2.5.3 --- Purification of PCR products --- p.44 / Chapter 2.5.4 --- Confirmation of PCR products --- p.45 / Chapter 2.5.5 --- cDNA probe labeling --- p.46 / Chapter 2.6 --- RNA extraction --- p.46 / Chapter 2.6.1 --- Extraction of total RNA from testicular cell lines --- p.46 / Chapter 2.6.2 --- Extraction total RNA from testicular tissues --- p.50 / Chapter 2.7 --- Northern blot analysis --- p.51 / Chapter 2.7.1 --- Measurement of total RNA concentration --- p.51 / Chapter 2.7.2 --- RNA gel electrophoresis --- p.52 / Chapter 2.7.3 --- Transfer of RNA to membrane --- p.53 / Chapter 2.7.4 --- Hybridization with [α-32P]dCTP-labelled probes --- p.53 / Chapter 2.7.5 --- Autoradiography and densitometric quantification --- p.54 / Chapter 2.8 --- Data and statistical analysis --- p.55 / Chapter 3. --- Results / Chapter 3.1 --- Effects of hypoxia and cobalt chloride treatment on VEGF expression in TM3 and TM4 cells --- p.57 / Chapter 3.2 --- Effects of testicular torsion on VEGF expression in adult rat testes --- p.61 / Chapter 3.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.66 / Chapter 3.4 --- Effect of cadmium on VEGF expression in TM3 and TM4 cells --- p.66 / Chapter 3.5 --- Effect of dexamethasone on Cd-induced increase in VEGF expression in TM3 cells --- p.73 / Chapter 3.6 --- Effect of cadmium treatment on VEGF expression in the adult rat testes --- p.73 / Chapter 3.7 --- Effect of Leydig cell depletion on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.8 --- Effect of dexamethasone on basal and Cd-induced expression of VEGF in adult rat testes --- p.76 / Chapter 3.9 --- "Effects of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.79 / Chapter 3.10 --- Effect of hCG on VEGF expression in MLTC-1 cells --- p.92 / Chapter 3.11 --- "Effect of EL-lα, IL-1β, IL-6, TNF- α and TNF- β on VEGF expression in TM3 cells" --- p.92 / Chapter 3.12 --- Effect of bFGF and TGF- β1 on VEGF expression in TM3 cells --- p.102 / Chapter 4. --- Discussion / Chapter 4.1 --- Upregulation of VEGF expression in TM3 and TM4 cells by hypoxia and cobalt chloride --- p.108 / Chapter 4.2 --- Effect of testicular torsion on VEGF expression in adult rat testes --- p.110 / Chapter 4.3 --- Antagonism of hypoxic induction of VEGF expression in TM3 cells by nitric oxide --- p.111 / Chapter 4.4 --- "Effect of cadmium on VEGF mRNA levels in TM3 and TM4 cells, and in adult rat testes" --- p.113 / Chapter 4.5 --- "Effect of hCG,forskolin and phorbol ester on VEGF expression in TM3 and TM4 cells" --- p.116 / Chapter 4.6 --- Effect of cytokines and growth factors on VEGF expression in TM3 cells --- p.119 / Chapter 5. --- References --- p.123

Acute myocardial infarction is the commonest cause of death in firefighters, accounting for 45% of all deaths on duty. Compared with an average life expectancy of 77 years in the general population, the average age of cardiovascular death in firefighters is 50 years suggesting that occupational hazards are responsible for premature disease. The risk of acute myocardial infarction is increased 12- to 136-fold during rescue and firefighting duties, and is likely to reflect a combination of factors including strenuous physical exertion, mental stress, heat and pollutant exposure. Previous studies have established that the duties of a firefighter, in particular fire suppression, put inordinate strain on the cardiovascular system yet the exact mechanisms underlying the increased risk of myocardial infarction remain poorly defined. In a series of studies, I assessed the effect of occupation-specific risk factors on cardiovascular health in a combination of controlled and real-life studies in order to better define these mechanisms, hypothesising that exposure to high temperatures, strenuous physical exertion, psychological stress and air pollution either alone or in combination caused vascular dysfunction and thrombosis. In order to assess if firefighters had a greater cumulative risk of cardiovascular disease due to their occupation at baseline, I assessed the cardiovascular function of group of healthy, off-duty firefighters and compared this to a group of healthy age- and sex-matched off-duty police officers; an occupational group with similar responsibilities but a much lower risk of on-duty cardiovascular events. I was able to demonstrate that traditional cardiovascular risk factors, vascular endothelial function and thrombogenicity were similar in the two groups concluding that the excess of cardiovascular events and deaths in on-duty firefighters are due to the acute and transient effects of strenuous physical exertion, psychological stress, heat and exposure to air pollutants. Having established that off-duty firefighters had no apparent increased risk of cardiovascular events, I then went on to clarify the effects of combustion derived air pollution in the form of wood smoke on the cardiovascular system. The suppression of wildland or forest fires is globally the single most important duty of the fire service. Previous work within our institution has demonstrated the adverse effects of combustion derived air pollution, in the form of diesel exhaust, on the cardiovascular system. In a similar fashion, I assessed the effect of a wood smoke inhalation in a group of healthy off-duty firefighters by performing controlled exposures to wood smoke utilising a unique and well characterised facility. Interestingly, unlike diesel-exhaust, the exposure to wood smoke had no adverse effect on vascular endothelial function or thrombogenicity in this group concluding that cardiovascular events during wildland fire suppression may not be directly related to wood smoke inhalation but instead precipitated by other mechanisms such as strenuous physical exertion or dehydration. Latterly, I proceeded to evaluate the effects of strenuous physical exertion and heat exposure by comprehensively assessing a number of cardiovascular end points following controlled exposure to a fire simulation activity in a group of healthy, off-duty firefighters. I was able to demonstrate that exposure to extreme heat and physical exertion impaired vasomotor function and increased thrombus formation. Moreover, I demonstrated cardiac troponin concentrations increased suggesting that fire suppression activity may cause myocardial injury. These important findings suggest pathogenic mechanisms to explain the association between fire suppression activity and acute myocardial infarction. In the final phase of work, I endeavoured to assess the effects of real-life firefighter activities on the cardiovascular system. In an ambitious study, I attempted to undertake a comprehensive assessment of cardiovascular function in healthy firefighters following three periods of duty: fire suppression, alarm response and non-emergency activity. I was unable to complete enough studies to adequately power an analysis and draw any firm conclusions about the effect of these duties on cardiovascular health. Further work is required in a real-world setting to more clearly define the occupational risk factors underlying the increased risk of cardiovascular events associated with specific firefighter duties Understanding the biological mechanisms and environmental factors that predispose firefighters to cardiovascular events is essential if we are to develop effective methods for the prevention of acute myocardial infarction on-duty. This body of work has greatly improved the understanding of the mechanisms underlying the increased risk of cardiovascular events on duty and calls for the immediate evaluation of current practice in order to minimise risk to firefighters in the future. Examples of where improvements should be made include strategies to ensure adequate hydration and cooling following exposure to heat and physical exertion, change to working patterns to limit the duration of extreme exposures, and education, training and screening programmes to reduce the impact of traditional and occupational cardiovascular risk factors.

acase@tulane.edu / An emerging need in the microcirculation research is the development of biomimetic angiogenesis models that recapitulate the complexity of a real tissue. Angiogenesis, defined as the growth of new vessels from pre-existing vessels, involves multiple cell types, such as endothelial and perivascular cells, in a multi-system setting since blood vessel networks are usually accompanied by lymphatic and nervous systems. Therefore, a need exists for a model of angiogenesis from intact microvascular networks that more closely reflects an in vivo scenario for the investigation of underlying mechanisms and the pre-clinical development of therapies. While other approaches have proven useful in identifying mechanistic signaling information, they are often limited in their complexity and capability to mimic physiologically relevant scenarios in one way or another and do not fully recapitulate the in vivo scenario. The first aim of this study was to demonstrate the ability for time-lapse comparisons of microvascular networks in angiogenesis scenarios to investigate the fate of vascular islands and investigate the endothelial cell plasticity. We developed a time-lapse angiogenesis model based on our previously introduced rat mesentery model. We demonstrated that time-lapse rat mesentery culture model is a powerful tool to study multi-cell, multi-system dynamics in microvascular networks. For the second aim of this study, we used the method developed in aim one to establish rat mesentery culture model as a novel anti-angiogenic drug screening tool. Using time-lapse model enabled tissue-specific comparisons before and after drug treatment to investigate its effects on entire microvascular networks. Validation of this method for anti-angiogenic drug testing was demonstrated using known angiogenesis inhibitor. Next, we showcased a potential application of the model for evaluating unknown effects of drug repositioning based on FDA-approved drug combinations. The results demonstrated the ability to identify concentration-dependent effects in an intact network scenario. The objective of the third aim was to showcase the capability of the rat mesentery culture model to study stem cell fate. We developed a protocol to deliver mesenchymal stem cells to mesentery tissues and culture for a period of time in a controlled environment. We confirmed the perivascular location of a subset of stem cells within capillaries, with morphologies resembling pericytes, and expressing pericyte markers. We also demonstrated that tracking stem cells within the microvascular networks is possible using the rat mesentery culture model. Furthermore, we reported a high variability in perivascular incorporation among cells from different donors. This work establishes for the first time, to the best of our knowledge, an ex vivo model to look at microvascular networks before and after growth. We confirmed, for the first time, vascular island incorporation as a new mode of angiogenesis using a novel method for time-lapse imaging of microvascular networks ex vivo. The results also establish this method for drug testing and stem cell tracking in a microvascular setting. / 1 / Mohammad Sadegh Azimi

No description available.

In vivo analyses of the Von Willebrand Factor (VWF) promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells. This fragment is active in all embryonic vessels of transgenic mice but in adult mice its activity is restricted to brain vascular endothelial cells, while endogenous VWF gene is expressed in vasculature of all major organs. In this study we demonstrate that a DNase I hypersensitive (HSS) sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. Our results demonstrated that Nuclear Factor 1 (NF1) and Nuclear transcription Factor Y (NFY) repressors contribute to VWF organ-specific regulation. Mutation of the NF1 binding site resulted in promoter activation in lung and heart, while mutation of the repressor corresponding to a novel binding site for NFY resulted in promoter activation in kidney vasculature. / Experimental Medicine

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

This study examined DNA methylation patterns at promoters of endothelial cell (EC)-enriched genes during differentiation of mouse ES cells towards the EC. We have previously shown that eNOS, CD31, VE-cadherin and vWF, which have an EC-enriched pattern of gene expression are differentially methylated between EC and vascular smooth muscle cells. Given that differential promoter DNA methylation is functionally important we asked when these distinct patterns are established. Using the hanging drop method to differentiate ES cells, followed by FACS, we isolated early (EB-day4 VEGFR2-positive) and late (EB-day7 CD31-positive) endothelial progenitor cells. Though current paradigms suggest that lineage-restricted genes are methylated in ES cells, we show heterogeneous promoter DNA methylation. We show DNA demethylation at the CD31 promoter in EB-day 7 CD31-positive cells. In contrast, the eNOS promoter is still heavily methylated in EB-day 7 CD31 positive cells compared with murine EC where there is no DNA methylation.

<p>The number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin &#945;5&#946;1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.</p> / Dissertation

Angiogenesis is a fundamental process in reproduction and wound healing. Angiogenesis is also indispensable for solid tumor growth and metastasis, and also associated with angiogenic diseases. Beta-endorphin (£]-EP), derived from its precursor pro-opiomelancortin (POMC), is well known for its role in nociception and immune regulation. However, the function of morphine and £]-EP during angiogenesis remains characterization. One previous study indicated that morphine inhibited the proliferation and hypoxia-induced vascular endothelial growth factor (VEGF) release of endothelial cells. Contrastingly, another report found that morphine via Ras/PI3k/MAPK/ERK signaling promotes the survival and angiogenesis in endothelial cells. Besides, endogenous opioid peptides stimulated angiogenesis in chicken allantoic membrane assay through opioid receptors. Thus, the function and mechanism of £]-EP and opioid receptors in angiogenesis are controversial. This study evaluated the culture effects of £]-EP and morphine on angiogenesis . It was found that £]-EP stimulated the proliferation, migration, and tube formation of endothelial cells in a dose-dependent manner. Morphine at a high dose inhibited the proliferation, migration, and tube formation of endothelial cells. In the ex vivo rat aortic ring assay, £]-EP enhanced, whereas morphine perturbed, the microvessel sprouting. We also confirmed the expression of MOR¡ADOR¡AKOR opioid receptor in endothelial cells. Application of naloxone, a selective opioid antagonist, and neutralizing antibodies of MOR abolished the angiogenic effect of £]-EP and morphine. Thus £]-EP and morphine exert the pro- and anti-angiogenic effect via MOR, respectively .Besides, £]-EP can be regarded as a novel angiogenic factor.

Angiogenesis is not only essential for tumor growth but is also implicated in invasion of the cancer cells into the circulation, and growth of dormant micro-metastases into frank metastatic lesions. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis as well as in solid tumors. It also has a role for VEGF in hematopoietic neoplasms; although has not been fully elucidated. This study will examine the VEGF secretary activity of malignant cells in the patients with hematologic malignancies. Supernatants of cell culture after 72 hours & 7th day were analyzed for VEGF value by ELISA method. The purposes of this study are to assay the VEGF activity and its correlation with disease prognosis in various hematological disorders; and to detect the VEGF autocrine activity of tumor cells in sequential culture without any stimulation in vitro. Results: our research samples were 75 specimens. The VEGF value was low in 13 cases of benign diseases, no obvious auto-secretary activity of those cells. There was no significant correlation between VEGF value and disease status in acute lymphoblastic leukemia; however, the cases were too small to had exact predict value (only 7 cases). In 17 Cases of malignant lymphoma, low D0 VEGF value (<300 pg) had good prognosis; those cases relapsed after treatment had high auto-secretary activity (high VEGF value of 7th culture day) and had bad disease prognosis, but there was no statistic significance. In 14 Cases of acute myelogenous leukemia, high D0 VEGF value (>150 pg) and high VEGF value of 7th culture day both presented bad disease outcome. In 16 cases of chronic myeloid leukemia (CML), low plasma VEGF level (D0<300pg) was all in chronic phase; all cases in accelerated phase (3 cases) and acute blastic crisis (5 cases) presented with high plasma VEGF level (>300pg). Patients with high plasma VEGF level had 70 % (7of 10) in worse disease status (P=0.010). Patients in chronic phase of CML had low VEGF auto-secretary activity and most of the blastic crisis patients were with high VEGF auto-secretary activity and also had bad prognosis. (P=0.248) Conclusion: although our study is a primary result, study cases are varied, but it still provide important information that VEGF has an important role in hematological malignancies. We will process further research of single and specific disease in the future to analyze the exact correlation of VEGF and hematological diseases.