Lun Samantha Wei Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 142-174). / Abstracts in English and Chinese. / Contents --- p.ii / Acknowledgements --- p.vii / Abstract --- p.viii / 摘要 --- p.xi / Chapter Section 1. --- Introduction / Chapter 1.1 --- General review of the epididymis --- p.1 / Chapter 1.1.1 --- Structure of the epididymis --- p.1 / Chapter 1.1.2 --- Function of the epididymis --- p.3 / Chapter 1.1.3 --- Regulation of the epididymal function --- p.5 / Chapter 1.2 --- Vascular endothelial growth factor (VEGF) --- p.8 / Chapter 1.2.1 --- VEGF peptides --- p.8 / Chapter 1.2.2 --- Biological activities of VEGF --- p.10 / Chapter 1.2.3 --- Hormonal regulation of VEGF --- p.11 / Chapter 1.3 --- VEGF receptors --- p.12 / Chapter 1.3.1 --- Flt-1 or VEGFR1 --- p.12 / Chapter 1.3.2 --- Flk-1 or VEGFR2 --- p.13 / Chapter 1.4 --- Caveolae --- p.15 / Chapter 1.4.1 --- Overview of caveolae --- p.15 / Chapter 1.4.2 --- Caveolins/caveolin-1 --- p.16 / Chapter 1.4.3 --- Caveolae and VEGF --- p.18 / Chapter 1.4.4 --- Caveolae and the epididymis --- p.20 / Chapter 1.5 --- VEGF/ VEGF receptors in the epididymis --- p.20 / Chapter 1.6 --- Aims of study --- p.22 / Chapter Section 2. --- Materials and Methods / Chapter 2.1 --- Materials --- p.24 / Chapter 2.2 --- Animal surgery --- p.35 / Chapter 2.2.1 --- Animals --- p.35 / Chapter 2.2.2 --- Castration and hemi-castration --- p.35 / Chapter 2.2.3 --- Efferent duct ligation (EDL) --- p.36 / Chapter 2.2.4 --- Tissue collection --- p.37 / Chapter 2.3 --- Epididymal cell culture --- p.38 / Chapter 2.4 --- Sample preparation --- p.40 / Chapter 2.4.1 --- Collection of epididymal plasma and sperm --- p.40 / Chapter 2.4.2 --- Purification of caveolae fraction --- p.41 / Chapter 2.5 --- Reverse-transcription polymerase chain reaction (RT-PCR) and semi-quantitative RT-PCR --- p.43 / Chapter 2.5.1 --- Preparation of RNA from epididymal tissues --- p.43 / Chapter 2.5.2 --- Quantitation of total RNA --- p.44 / Chapter 2.5.3 --- Reverse transcription (RT) and polymerase chain reaction (PCR) --- p.44 / Chapter 2.5.4 --- Purification and authenticity confirmation of PCR products --- p.50 / Chapter 2.6 --- Western immunoblotting --- p.53 / Chapter 2.6.1 --- Preparation of protein --- p.53 / Chapter 2.6.2 --- SDS-PAGE --- p.53 / Chapter 2.6.3 --- Western immunoblotting --- p.55 / Chapter 2.7 --- Immunohistochemistry --- p.56 / Chapter 2.7.1 --- Preparation of tissue sections --- p.56 / Chapter 2.7.2 --- Immunohistochemical staining of tissue sections --- p.57 / Chapter 2.7.3 --- Immunostaining of cultured cells --- p.59 / Chapter 2.8 --- Enzyme Linked Immunosorbant Assay (ELISA) --- p.59 / Chapter 2.9 --- Statistical analyses --- p.60 / Chapter Section 3. --- Results / Chapter 3.1 --- Expression and localization of VEGF in the rat epididymis --- p.62 / Chapter 3.1.1 --- RT-PCR of VEGF in the rat epididymis --- p.62 / Chapter 3.1.2 --- Western immunoblot of VEGF in the rat epididymis --- p.63 / Chapter 3.1.3 --- Developmental changes in VEGF expression in the rat epididymis --- p.66 / Chapter 3.1.4 --- Immunolocalization of VEGF in the rat epididymis --- p.66 / Chapter 3.1.5 --- Summary of the localization and expression of VEGF in the rat epididymis --- p.72 / Chapter 3.2 --- Expression and localization of VEGF receptors in the rat epididymis --- p.74 / Chapter 3.2.1 --- RT-PCR of Flt-1 and sFlt-1 in the rat epididymis --- p.74 / Chapter 3.2.2 --- Western immunoblot of Flt-1 in the rat epididymis --- p.74 / Chapter 3.2.3 --- Immunolocalization of Flt-1 in the rat epididymis --- p.75 / Chapter 3.2.4 --- RT-PCR of Flk-1 in the rat epididymis --- p.75 / Chapter 3.2.5 --- Western immunoblot of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.6 --- Immunolocalization of Flk-1 in the rat epididymis --- p.79 / Chapter 3.2.7 --- Summary on the localization and expression of VEGF receptors in the rat epididymis --- p.83 / Chapter 3.3 --- Detection of VEGF immunoreactivity in epididymal plasma and sperm lysate collected from cauda epididymidis --- p.83 / Chapter 3.4 --- Effect of castration on VEGF and VEGF receptor expression in the rat epididymis --- p.84 / Chapter 3.4.1 --- Effect of castration with or without testosterone replacement on VEGF expression in the rat epididymis --- p.84 / Chapter 3.4.2 --- Effect of castration with or without testosterone replacement on Flt-1 expression in the rat epididymis --- p.94 / Chapter 3.4.3 --- Effect of castration with or without testosterone replacement on Flk-1 expression in the rat epididymis --- p.98 / Chapter 3.5 --- Effect of efferent duct ligation and hemicastration on VEGF peptide levels in the rat epididymis --- p.102 / Chapter 3.5.1 --- Effect of efferent duct ligation on VEGF expression in the rat epididymis --- p.102 / Chapter 3.5.2 --- Effect of hemi-castration on VEGF expression in the rat epididymis --- p.106 / Chapter 3.6 --- "Summary on the effects of castration, efferent duct ligation, and hemicastration on the epididymal weight, and VEGF/VEGF receptor expression in the rat epididymis" --- p.107 / Chapter 3.6.1 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on the epididymal weight" --- p.107 / Chapter 3.6.2 --- "Summary on the effects of castration, efferent duct ligation and hemicastration on VEGF and VEGF receptor expression in the rat epididymis" --- p.112 / Chapter 3.7 --- Localization and expression of caveolin-1 and 226}0ؤ2 in the rat epididymis --- p.113 / Chapter 3.7.1 --- RT-PCR of caveolin-1 and caveolin-2 in the rat epididymis --- p.113 / Chapter 3.7.2 --- Western immunoblot of caveolin-1 and caveolin-2 in the rat epididymis --- p.114 / Chapter 3.7.3 --- Immunolocalization of caveolin-1 in the rat epididymis --- p.117 / Chapter 3.7.4 --- Summary on the localization and expression of caveolin-1 and -2 in the rat epididymis --- p.119 / Chapter 3.8 --- Co-localization of VEGF receptors with caveolae in the rat epididymis --- p.119 / Chapter Section 4. --- Discussion --- p.124 / Chapter 4.1 --- VEGF expression and localization --- p.124 / Chapter 4.2 --- VEGF receptors expression and localization --- p.129 / Chapter 4.3 --- Possible VEGF action in the rat epididymis --- p.133 / Chapter 4.4 --- Regulation of VEGF and its receptor expression by androgen and/or other testicular factors --- p.136 / References

Both VEGF and its receptor proteins were localized by immunostaining technique in the luminal epithelium, smooth muscle cells and blood vessels within the oviduct. Moreover, by means of semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) techniques, it has been demonstrated that mRNA of VEGF and its receptors in both healthy and diseased oviduct is expressed preferentially at the time and place where the amount of oviduct fluid is prominent. This supports the notion that VEGF may be a regulator of oviductal secretion. This thesis has consistently demonstrated a modulation pattern of flt-1 expression that is similar to its ligand VEGF in both physiological and pathological conditions. This suggests that flt-1 may be the main receptor responsible for the action of VEGF in the oviduct. As illustrated in both the in-vivo and in-vitro models, the expression of VEGF and flt-1 in the human oviduct is stimulated directly by gonadotropins without the influence of ovarian sex hormones. / Increased knowledge on the regulatory mechanisms of oviductal fluid formation, the first environment that human embryos are exposed to, will be valuable from the clinical management point of view. / Oviductal fluid is a complex mixture of plasma-derived constituents and proteins synthesized by the oviduct epithelium. It has been postulated that vascular endothelial growth factor (VEGF), a known permeability promoter, may be an important regulator of oviductal fluid secretion by stimulating vascular permeability and so serum transudation. However, little is known about the expression of VEGF in the human oviduct. This thesis investigated the modulation of VEGF and its receptors (flt-1 and KDR) in the healthy oviducts, from fertile women undergoing tubal sterilization for unwanted fertility or hysterectomy for benign gynecological conditions, as well as in the hydrosalpinges from sterile women undergoing salpingectomy before the treatment of in-vitro fertilization and embryo transfer. / Lam Po Mui. / Adviser: Christopher J. Haines. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (M.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 147-179). / 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.

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.

<p>Synthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs. </p><p>This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.</p> / Dissertation

The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis, abdominal aortic aneurysms, and heart valve disease in regions exposed to disturbed flow leading to low or oscillatory shear stress at the wall of the blood vessel or the surface of the valve leaflet. The novel and significant finding of this study is that mouse aortic endothelial cell exposure to pro-atherogenic oscillatory shear stress (OS) (+/- 5 dynes/cm2) increased their production of cathepsins, the family of lysosomal cysteine proteases that are potent elastases and collagenases leading to protease degradation and remodeling of the extracellular matrix structural components. Conversely, atheroprotective unidirectional laminar shear stress (LS) (15 dynes/cm2) decreased elastase and gelatinase activities of endothelial cells through a shear stress mediated reduction in cathepsins K, L, and S activity. Their endogenous inhibitor, cystatin C, was found to be inversely regulated by shear stress; LS increased its secretion by endothelial cells while OS decreased it. Binding of free cystatin C in the conditioned media to carboxymethylated papain coated agarose beads led to an increase in cathepsin activity since the available cathepsin was not inhibited. To verify these findings in human samples, immunohistochemical analysis of cystatin C and cathepsin K was performed on human coronary arteries. Cathepsin K stained strongly in the endothelial layer of vessels with degraded internal elastic lamina while cystatin C staining intensity was strongest overlying minimally diseased vessels. Additional roles for cathepsins K, L, and S were found in endothelial cell alignment in response to unidirectional laminar shear stress, endothelial cell migration, and programmed cell death. We conclude that there is an inverse regulation of cathepsins and cystatin C in endothelial cells by LS and OS and identify the cathepsin family of proteases as potential targets for therapeutic intervention of cardiovascular disease development at sites of disturbed flow.

Non-hematopoietic stromal cells (SCs) promote and regulate adaptive immunity through numerous direct and indirect mechanisms. SCs construct and support the secondary lymphoid organs (SLOs) in which lymphocytes crawl on stromal networks and inspect antigen-presenting cells for surface-display of cognate antigens. SCs also secrete survival factors and chemotactic cues that recruit, organize, and facilitate interactions among these leukocytes. They influence antigen access by secreting and ensheathing extracellular matrix-based conduit networks that rapidly convey small, soluble lymph-borne molecules to the SLO core. Furthermore, lymph node stromal cells (LNSCs) directly induce \(CD8^+\) T cell tolerance to peripheral tissue restricted antigens and constrain the proliferation of newly activated T cells in these sites. Thus, stromal-hematopoietic interactions are crucial for the normal functioning of the immune system. LNSCs are extremely rare and difficult to isolate, hampering the thorough study of their biology. In order to better understand these stromal subsets, we sorted fibroblastic reticular cells (FRCs), lymphatic endothelial cells, blood endothelial cells, and podoplanin \(^−CD31^−\) cells (double negative stromal cells; DNCs) to high purity from resting and inflamed murine lymph nodes. We meticulously analyzed the transcriptional profiles of these freshly isolated LNSCs as part of the Immunological Genome Project Consortium. Analysis of the transcriptional profiles of these LNSC subsets indicated that SCs express key immune mediators and growth factors, and provided important insights into the lymph node conduit network, FRC-specialization, and the DNC identity. Examination of hematopoietic and stromal transcription of ligands and cognate receptors suggested complex crosstalk among these populations. Interestingly, FRCs dominated cytokine and chemokine transcription among LNSCs, and were also enriched for higher expression of these genes when compared with skin and thymic fibroblasts, consistent with FRC-specialization. LNSCs that were isolated from inflamed lymph nodes robustly upregulated expression of genes encoding cytokines, chemokines, antigen-processing and presentation machinery, and acute-phase response molecules. Little-explored DNCs showed many transcriptional similarities to FRCs, but importantly did not transcribe interleukin-7. We identified DNCs as consisting largely of myofibroblastic pericytes that express integrin \(\alpha 7\). Together these data comprehensively describe the transcriptional characteristics of four major LNSC subsets isolated from resting and inflamed SLOs, offering many avenues for future study.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.