ABSTRACT: UTILIZING NOVEL DOSE EQUIVALENCE METHODOLOGIES TO EXAMINE COCAINE'S EFFECTS ON THE VASCULATURE Neil S. Lamarre Doctor of Philosophy Temple University School of Medicine, 2013 Doctoral Advisory Committee Chair: Ronald J. Tallarida, Ph.D. Cocaine abuse and addiction is a serious health problem, resulting in thousands of emergency room visits and deaths each year in the United States. It is particularly toxic to the cardiovascular system, including deleterious effects on the peripheral vasculature. These effects are not well understood, but evidence suggests chronic cocaine use may lead to endothelial dysfunction, thereby increasing relative risk of a number of other cardiovascular diseases including stroke, aneurysm, myocardial infarction, hypertension, etc. Data from our lab, and others, suggest that the presence of a functional endothelium has a dramatic effect on the contractility of the rat aorta that is agonist-specific. Attenuation of this endothelium-dependent vasodilatory component of agonist action is a primary feature of endothelial dysfunction. We have utilized dose equivalence theory to calculate the dose response relationship for the endothelium-dependent vasodilatory component of an agonist causing overt vasoconstriction. This component cannot be measured directly, but our novel methodology allows us to quantitate agonist-specific impairment of vasodilation, and describe it using the familiar parameters of the dose response curve. Another strength of this method, relative to currently used in vitro methods, is that it also avoids the confounding variable of a second agonist used to produce the initial vasoconstriction. To validate the methodology, a pilot study was performed examining the endothelial dysfunction in STZ-induced diabetic rats, as a positive control for endothelial dysfunction. Interestingly, this treatment showed impairment in the endothelium-dependent vasodilatory component of action of norepinephrine, but not of angiotensin-II. Thus, our initial hypothesis was confirmed - that disruption of the vasodilatory components of various agonists are independent, and that agonist-specific information may prove useful. Next, we employed our new methodology utilizing the rat aorta as our vascular model to test the hypothesis that chronic cocaine administration causes endothelial dysfunction. We first examined the endothelium-dependent vasodilation component of a number of physiologically important vasoconstrictors, and attempted to determine which vasodilatory mediators contributed to the effect. We found the endothelium to have a profound effect on the dose response curve to three important endogenous agonists. These data suggest that under conditions of endothelial dysfunction exaggerated vasoconstriction could occur, even within normal plasma concentration ranges of these vasoconstrictors, resulting in elevated blood pressure and further damage to the endothelium over time. No endothelial dysfunction was observed with this treatment paradigm, using our methodology or the standard approach. This may be a result of insufficient duration of cocaine treatment, or a result of our selection of the rat aorta as a model. We wanted to further investigate which vasodilatory mechanisms were involved in this vasodilatory component of action. We inhibiting various endothelium-derived mediators of this vasodilatory component of action (such as nitric oxide or prostacyclin), which revealed differential activation of these mediators by the agonists examined. For example, inhibition of nitric oxide synthesis abolished the endothelium-dependent vasodilatory component of endothelin-1, but only partially attenuated that of angiotensin-II. Thus, the agonist-specific pattern of impairment may also prove useful in examining the underlying mechanisms of impaired vasodilation. Endothelial dysfunction is one reported consequence of long term cocaine abuse; however, there are conflicting reports on the acute vascular effects of cocaine, with some reports concluding that cocaine is a vasoconstrictor, and some reporting its action as a vasodilator. There are in vitro reports of cocaine causing release of vasoconstrictors from the endothelium, which supports the longstanding notion of cocaine as a vasoconstrictor. However, one recent report demonstrates a dose-dependent vasodilatory effect of cocaine in rat aorta that is independent of the endothelium. This complexity is perhaps due, in part, to cocaine's affinity for a number of molecular targets, acting in combination. In examining the acute action of cocaine in our preparation, we observed an "inverted-U" shaped dose response, also referred to as a hormetic dose response curve. We then applied dose equivalence methodology in order to derive the "unknown" second component contributing the vasodilatory action of cocaine at higher doses. This methodology lets us calculate this unknown component, and describe it with the familiar parameters of a dose response curve, which could potentially aid in the identification of the unknown component. The preliminary studies with acute cocaine utilized a sub-maximal dose of phenylephrine in order to observe tension changes in either direction. This prompted us to further characterize the interaction of cocaine with other alpha adrenoceptor agonists. Importantly, because cocaine alone had no effect at doses up to 100 µM, but potentiated the vasoconstriction of alpha agonists, the interaction is therefore synergistic. This constitutes evidence of a previously undescribed mechanism contributing to cocaine's vasoconstricting effect. In vivo, reuptake inhibition is a major mechanism for cocaine-induced vasoconstriction, but is excluded in this experiment by virtue of low levels of sympathetic innervation in the rat aorta, and the use of methoxamine, an alpha agonist not subject to the reuptake mechanisms. This interaction may contribute to cocaine-induced vasoconstriction in the coronary arteries, especially in circumstances of endothelial dysfunction. In summary, the work presented in this dissertation applies new methodologies utilizing dose equivalence theory to the study of cocaine's effects on peripheral vasculature, and presents novel findings of synergy with respect to cocaine's enhancement on the action of alpha adrenoceptor-mediated vasoconstriction. / Pharmacology

Cardiovascular disease and the associated endothelial dysfunction are characterized by leukocyte activation, decrease endothelial nitric oxide synthase (eNOS) activity, and increased endothelial cell adhesion molecules expression. This leads to the release of myeloperoxidase (MPO) by activated neutrophils and monocytes. MPO is a peroxidase enzyme that plays an important role in innate immune host defense, however it has been shown to play a pathogenic role in cardiovascular disease, mainly by causing endothelial dysfunction. The molecular mechanisms through which MPO induces endothelial damage are not fully understood. Calpains are a family of calcium-dependent proteases. Two calpain isoforms, µ- and m-calpain, are expressed in the vascular wall, including endothelial cells. Activation of calpains has been recently implicated in inflammatory disorders of the vasculature. The goal of this study was to test the hypothesis of a role for calpains in the molecular mechanism(s) through which MPO causes endothelial dysfunction and vascular inflammation. To explore if MPO activates calpain and to identify the calpain isoform(s) involved, we first studied the effects of MPO treatment on calpain activity in mouse lung microvascular endothelial cells (MMVEC). MMVECs were stimulated with 10 nmol/L MPO for 60, 120, 180, and 240 min. Using a fluorescent calpain activity assay, we found that MPO time dependently activates calpain in endothelial cells, with peak activity reached within 180 min. Using immunoblot analysis techniques we demonstrated that the calpain isoform targeted by MPO is µ-calpain. Interestingly, using a biotin switch assay,10 nmol/L MPO appears to activate the µ-calpain isoform via denitrosylation of its C-terminus domain. Using MMVECs, we studied the effects of the MPO/µ-calpain signaling on endothelial dysfunction. MMVECs were stimulated with 10 nmol/L MPO for 180 min. Expression levels of Protein Phosphatase 2 (PP2A), total 5' AMP-activated protein kinase (AMPK), Thr172 phospho-AMPK, total endothelial nitric oxide synthase (eNOS),Ser1177 phospho-eNOS, total protein kinase B (AKT), Ser473 phospho AKT, Adiponectin receptor 1 (AdipoR1), and Adiponectin receptor 2 (AdipoR2), were measured by immunoblot analysis. Interestingly, MPO impaired Thr172AMPK, Ser1177eNOS, but not Ser 473 AKT phosphorylation in a calpain dependent manner. On the other hand, MPO significantly increased the expression levels of PP2A. Inhibition of PP2A with okadiak acid decreased the phosphorylation levels of AMPK, and eNOS, indicating that PP2A is a downstream target of the MPO/calpain system. MPO treatment significantly increased the expression of vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells. Pharmacological inhibition of calpain activity attenuated expression of VCAM-1. MPO also decreased protein levels of AdipoR1, and AdipoR2 in a calpain dependent manner. The treatment of MMVEC with adiponectin in the presence of MPO was not able to restore AdipoR2 expression levels. Using genetically modified mice, we found evidence of reduced leukocyte adhesion to the aortic endothelium in response to MPO in µ-calpain deficient mice, compared to wild-type mice . These effects appears to be attributed to the endothelial calpain, since incubating wild type aortas with calpain deficient leukocytes did not reduce leukocyte adhesion to the endothelium. The data in this study first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO, with MPO activating the µ-calpain isoform via denitrosylation. Our data also report that increased calpain activity downregulats the expression of a number of signaling molecules important for endothelial cell function. This study may provide the MPO/calpain/PP2A signaling pathway as a novel pharmacological targets for the treatment of inflammation-driven vascular disorders. / Biomedical Sciences

The current study evaluated the potentially detrimental effects of daily resistance training on cardiovascular health using a longitudinal study design. This study also addressed the effects of resistance training on vascular endothelial function. Recent cross-sectional studies have shown resistance trained individuals have reduced whole-body arterial compliance compared to sedentary controls and that the age-associated reduction of arterial compliance is augmented in resistance trained athletes. The effect of resistance training on vascular endothelial function has not been addressed to date in the literature. Twenty-eight young healthy males (age: 23±3.9 [mean±SD]) were whole body resistance trained five times a week for twelve weeks, using a split body design. Measurements of supine resting arterial blood pressure at the brachial artery, carotid, brachial and femoral cross-sectional compliance, and brachial vascular endothelial function (using flow-mediated dilation) were acquired prior to, halfway through and following the exercise training protocol. Strength of various body segments increased significantly following the resistance training program. Shoulder press one repetition maximum (1RM) lifts increased from 141.4±7.6 lbs. to 185.2±8.8 lbs. and double leg press 1RM from 483.0±29.0 lbs. to 859.8±52.1 lbs. Resting diastolic blood pressure increased significantly from Mid to Post training (61.8±1.3 mmHg to 65.4±1.2 mmHg) yet was not significantly changed from Pre values (62.9±1.2 mmHg). Pulse pressure was reduced significantly with exercise training by the Post training time-point (Pre 63.3±1.9 mmHg; Mid 59.0±2.4 mmHg; Post 53.7±2.8 mmHg). Mean arterial carotid and femoral artery diameters were not changed with resistance training; however, mean brachial artery diameter increased by the Mid training time-point and remained elevated at the Post training time-point (Pre 3.81±0.10 mm; Mid 4.03±0.1 0 mm; Post 4.04±0.11 mm). Cross-sectional compliance did not change at the carotid or the brachial arteries, however the femoral artery experienced a reduction of compliance by the Mid time-point that remained to the Post training time-point (Pre 0.162±0.012 mm²/mmHg; 0.125±0.013 mm²/mmHg; Post 0.129±0.015 mm²/mmHg). Brachial vascular endothelial function measured using flow-mediated dilation did not show a significant change with resistance training. When normalized for shear rate (which was also unaltered with resistance training) there were no changes in endothelial function. Peak and 1 0-s average brachial post-occlusion blood flow was enhanced with resistance training (Pre 247.5±14.0 ml/min; Mid 331.1±18.5 ml/min; Post 290.5±21.0 ml/min) possibly revealing enhanced resistance vessel function. In conclusion, resistance exercise training results decreased PP, reduced femoral compliance, an increase in mean brachial artery diameter and enhanced post-ischemic blood flow. The exact mechanisms responsible for such changes remain unknown and require further investigation. / Thesis / Master of Science (MS)

Diabetic retinopathy (DR) is an incurable, chronic disease that is the leading cause of blindness in working-age adults. A prominent characteristic of DR is the extensive dysfunction within the retina microvasculature. Specialized vascular cells known as pericytes (PCs) are lost or become dysfunctional during disease progression; a thickening of the extracellular matrix (ECM) composing the vascular basement membrane (vBM) and endothelial cell (EC) tight junction disruption are also key features of this disease and contribute to its pathogenesis. PC loss is believed to be a central cue for disease initiation. However, studies inducing PC loss and observing acute changes in the vasculature did not report severe vessel damage or vBM thickening, suggesting that the effects of PC loss occur over a longer period of time. Because the chronic effects of PC loss are more difficult to ascertain, especially in a complex condition such as DR, the mechanisms underlying microvascular defects in DR remain poorly understood. The work presented in this dissertation focuses on pericyte-endothelial cell interactions and their interplay with the ECM/vBM during a variety of physiological and pathological conditions. First, we isolated and functionally validated a primary mouse embryonic PC cell line that we then applied to a co-culture model with ECs to better understand the dynamic interactions between these two critical components of the capillary wall. In the co-culture model, we found that primary PCs promoted EC organization into vessel-like structures and enhanced EC-EC junctions. To complement these in vitro studies, we analyzed animal models and human tissue for the PC-EC interactions and ECM/vBM remodeling under different conditions (physiological and pathological). Moreover, we analyzed microglia and astrocytes to enhance our understanding of the tissue-vessel interface, bolstering our experimental results and facilitating the generation of more hypotheses for future research. Overall, our work suggests that PC-EC interactions in diabetic scenarios play a crucial role in ECM/vBM remodeling; engagement with the ECM/vBM in turn impacted PC behaviors including migration away from the endothelium and induced EC loss of tight junctions, key changes in the onset and progression of DR. / Doctor of Philosophy / Diabetic retinopathy is a group of eye diseases occurring in patients suffering from diabetes and is the leading cause of adult blindness among the working-aged. About one in three people with diabetes over the age of 40 have overt signs of DR. The primary cause for this disease is long-term, high blood sugar levels that damages blood vessels systemically as well as in the eye. Current treatments for DR can prevent the condition from getting worse, but no treatment exists that results in a complete cure. This work described in this dissertation focuses on the interactions between vascular pericytes and endothelial cells, two of the main cell types that compose capillaries (i.e. the smallest blood vessels important for oxygen delivery). The studies presented herein also focus on the response of these cells to the extracellular matrix, a scaffold of proteins that surround pericytes and endothelial cells to stabilize blood vessels. We found that extracellular matrix components dramatically increase as a result of the interactions between pericytes and endothelial cells exposed to diabetic conditions. These changes in the extracellular matrix also had important effects on pericytes and endothelial cells and their engagement with their environment and other cells. Taken together, our work suggests that pericyte-endothelial cell interactions and their crosstalk with the ECM play an important role in blood vessel formation and in the accumulation of microvascular defects that fuel diabetic retinopathy progression.

Yes / Interleukin-36 cytokines are predominantly expressed by epithelial cells. Significant upregulation of epidermal IL-36 is now a recognised characteristic of psoriatic skin inflammation. IL-36 is known to induce inflammatory responses in dendritic cells, fibroblasts and epithelial cells. Although vascular alterations are a hallmark of psoriatic lesions and dermal endothelial cells are well known to play a critical role in skin inflammation, the effects of IL-36 on endothelial cells are unexplored. We here show that endothelial cells including dermal microvascular cells express a functionally active IL-36 receptor. Adhesion molecules VCAM-1 and ICAM-1 are upregulated by IL-36γ stimulation and this is reversed by the presence of the endogenous IL-36 receptor antagonist. IL-36γ stimulated endothelial cells secrete the proinflammatory chemokines IL-8, CCL2 and CCL20. Chemotaxis assays showed increased migration of T cells following IL-36γ stimulation of endothelial cells. These results suggest a role for IL-36γ in the dermal vascular compartment and it is likely to enhance psoriatic skin inflammation by activating endothelial cells and promoting leukocyte recruitment.

Nitric oxide (NO), a vasoprotective and ubiquitous signaling molecule generated from the endothelial cells (EC) by the enzyme endothelial nitric oxide synthase (eNOS) have a vital role in regulation of vascular function and integrity. However, a significant attenuation of eNOS and NO leads to endothelial dysfunction (ED) and increased risk of cardiovascular disease (CVD) in diabetes. Lipoproteins particularly LDL, undergo glycation in diabetic patients and turns it into pro-atherogenic glycated LDL (glyLDL). However, the impact of glyLDL on eNOS, the transmembrane signalling events, involvement of mitochondrial and endoplasmic reticulum (ER) stress in EC remains unclear. Also, literatures reveal impaired platelet mitochondrial function in diabetes patients; however, the impact of family history of diabetes on platelet mitochondrial bioenergetics still remains unknown. In the present study, we had provided the evidence for diabetes-associated metabolic stress involving glyLDL can attenuate eNOS protein, gene and activity in EC, as well as glyLDL and high glucose attenuates eNOS content in EC. Receptor of advanced glycation end products (RAGE) and H-Ras pathway are implicated in the upstream signalling events in the downregulation of eNOS in EC. In addition, ER stress, impaired mitochondrial function due to significant reduction of complex-specific oxygen consumption and bioenergetics were identified in glyLDL-treated EC. Further, we have also detected significant impairment in platelet mitochondrial bioenergetics in healthy individuals with familial history of diabetes. Identifying the mechanisms involved in diabetes associated metabolic stress induced signaling in EC and early detection of mitochondrial impairment in healthy individuals will help to find new targets for the prevention and treatment of diabetic cardiovascular complications and improve quality of life in diabetic patients. / May 2015

<p>Endothelial dysfunction is the predominant pathophysiological state prior to the onset of atherosclerosis. Currently, treatments for endothelial dysfunction are evaluated in vitro using two-dimensional (2D) cell culture assays or in vivo animal models. Microphysiological systems are small-scale three-dimensional (3D) tissue models that recapitulate the native tissue structure and function. An ideal microphysiological system is comprised of human cells embedded within a 3D matrix introduced to physiological fluid perfusion. Immune challenge in the form of cytokines or immune cells further recapitulates the native microenvironment.</p><p>A vascular microphysiological system was developed from a small-diameter tissue engineered blood vessel (TEBV) in a perfusion culture circuit. TEBVs were created from collagen gels embedded with human neonatal dermal fibroblasts and plastically compressed to yield collagen constructs with high fiber densities. TEBVs are rapidly producible and can be directly introduced into perfusion culture immediately after fabrication. Endothelium-independent vasoconstriction in response to phenylephrine and endothelium-dependent vasodilation in response to acetylcholine were used to analyze the health and function of the endothelium non-destructively over time.</p><p>Endothelial dysfunction was induced through introduction of the pro-inflammatory cytokine tumor necrosis factor – α (TNF-α). Late-outgrowth endothelial progenitor cells derived from the peripheral blood of coronary artery disease patients (CAD EPCs) were evaluated as a potential endothelial source for autologous implantation in both a two-dimensional (2D) direct co-culture model as well as a 3D model as an endothelial source for a tissue engineered blood vessel. CAD EPCs demonstrated similar adhesive properties to a confluent, quiescent layer of smooth muscle compared to human aortic endothelial cells. Within the TEBV system, CAD EPCs demonstrated the capacity to elicit endothelium-dependent vasodilation. CAD EPCs were compared to adult EPCs from young, healthy volunteers. Both CAD EPCs and healthy volunteer EPCs demonstrated similar endothelium-dependent vasoactivity in response to acetylcholine; however, in response to TNF-α, CAD EPCs demonstrated a reduced response to phenylephrine at high doses.</p><p>The treatment of TEBVs with statins was explored to model the drug response within the system. TEBVs were treated with lovastatin, atorvastatin, and rosuvastatin for three days prior to exposure to TNF-α. In all three cases, statins prevented TNF-α induced vasoconstriction in response to acetylcholine within the TEBVs, compared to TEBVs not treated with statins. Overall, this work characterizes and validates a novel vascular microphysiological system that can be tested in situ in order to determine the effects of various patient populations and drugs on endothelial health and function under healthy and inflammatory conditions.</p> / Dissertation

Wong Chun Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 149-160). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Abbreviations --- p.v / Acknowledgement --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General review of angiogenesis --- p.1 / Chapter 1.1.1 --- Angiogenesis in development and growth --- p.1 / Chapter 1.1.2 --- The process of angiogenesis --- p.2 / Chapter 1.1.3 --- Types of factors controlling angiogenesis --- p.3 / Chapter 1.2 --- Roles of VEGF and its receptors in the regulation of angiogenesis --- p.6 / Chapter 1.2.1 --- VEGF --- p.6 / Chapter 1.2.2 --- VEGF receptors --- p.8 / Chapter 1.2.3 --- Regulation of VEGF expression by hypoxia and nitric oxide… --- p.10 / Chapter 1.2.4 --- Signal transduction mechanisms of VEGFR-1 and VEGFR-2 --- p.12 / Chapter 1.2.5 --- Anti-apoptotic effect ofVEGF on endothelial cells as a result of signal transduction of VEGFR-2 --- p.14 / Chapter 1.3 --- Angiopoietins --- p.15 / Chapter 1.3.1 --- Angiopoietin 1 (Ang-1) --- p.16 / Chapter 1.3.2 --- Angiopoietin 2 (Ang-2) --- p.19 / Chapter 1.3.3 --- Angiopoietins 3 and 4 (Ang-3 and Ang-4) --- p.24 / Chapter 1.4 --- "Interaction among VEGF, angiopoietin and Tie in the maintenance of vasculature" --- p.25 / Chapter 1.5 --- Tyrosine kinase with immunoglobulin and EGF factor homology domains - Tie 1 and Tie 2 --- p.28 / Chapter 1.6 --- Angiopoietin expression in female reproductive tissues (ovary) --- p.33 / Chapter 1.7 --- Testicular angiogenesis --- p.37 / Chapter 1.8 --- Aims of the present study --- p.38 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Preparation of primary cells from rat testes --- p.40 / Chapter 2.1.1 --- Sertoli cell preparation --- p.40 / Chapter 2.1.2 --- Germ cell preparation --- p.41 / Chapter 2.1.3 --- Interstitial cell and Leydig cell preparation --- p.43 / Chapter 2.2 --- Cell cultures --- p.45 / Chapter 2.2.1 --- Reagents and cell lines --- p.45 / Chapter 2.2.2 --- Cell lines of mouse TM3 Leydig cells and TM4 Sertoli cells --- p.45 / Chapter 2.2.3 --- Mouse MLTC-1 Leydig tumour cells --- p.46 / Chapter 2.2.4 --- Rat R2C Leydig tumour cells --- p.46 / Chapter 2.2.5 --- Rat LC540 Leydig tumour cells --- p.47 / Chapter 2.2.6 --- "Rat C6 glioma cells.............," --- p.47 / Chapter 2.3 --- "Analyses of Angiopoietin 1, Angiopoietin 2, Angiopoietin3, Tie 1 receptor, and Tie 2 receptor mRNA in testicular cell lines and testicular tissues" --- p.48 / Chapter 2.3.1 --- Extraction of total RNA from testicular cell lines and testicular tissues --- p.48 / Chapter 2.3.2 --- Quantitation of total RNA --- p.50 / Chapter 2.3.3 --- First strand cDNA synthesis by reverse transcription (RT) --- p.51 / Chapter 2.3.4 --- Normalization of the amounts of cDNA usedin polymerase chain reaction (PCR) --- p.52 / Chapter 2.3.5 --- Polymerase chain reaction (PCR) --- p.53 / Chapter 2.3.6 --- Purification of PCR products --- p.65 / Chapter 2.3.7 --- Confirmation of PCR product authenticity by automated DNA sequencing --- p.66 / Chapter 2.4 --- Western blot analysis --- p.68 / Chapter 2.4.1 --- Preparation of cell lysates from primary testicular cells and testicular cell lines --- p.68 / Chapter 2.4.2 --- Preparation of mouse testicular tissue and adult rat testicular tissue lysates --- p.68 / Chapter 2.4.3 --- Determination of protein concentration --- p.69 / Chapter 2.4.4 --- Reagents for Western blot analysis --- p.70 / Chapter 2.4.5 --- Preparation of protein samples and markers for Western blot analysis --- p.71 / Chapter 2.4.6 --- Sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.72 / Chapter 2.4.7 --- Transfer of proteins to membrane --- p.74 / Chapter 2.4.8 --- Blocking of the membrane --- p.74 / Chapter 2.4.9 --- Immunoblotting --- p.75 / Chapter 2.5 --- "Immunohistochemical staining for Ang-1, Ang-2，Ang-3, Tie 1 and Tie 2 in rat testes" --- p.78 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Expression of Ang-1 and Ang-1 alternatively spliced transcripts in the testis and other testicular cell types --- p.81 / Chapter 3.1.1 --- Detection of Ang-1 expression in the testis and and testicular cell types by nested PCR --- p.81 / Chapter 3.1.2 --- Detection of Ang-1 expression in testicular cell lines by nested PCR --- p.82 / Chapter 3.1.3 --- Sequence analysis of Ang-1 transcript amplified from adult rat testis --- p.84 / Chapter 3.1.4 --- Detection of alternatively spliced species of Ang-1 mRNA in the testis and other testicular cell lines --- p.87 / Chapter 3.2 --- Expression of Ang-2 and Ang-2 isoforms in the testis and various testicular cell types --- p.94 / Chapter 3.2.1 --- Detection of Ang-2 expression in the testis and testicular cell types by nested PCR --- p.94 / Chapter 3.2.2 --- Detection of Ang-2 expression in testicular cell lines by nested PCR --- p.96 / Chapter 3.2.3 --- Sequence analysis of Ang-2 transcript amplified from adult rat testis --- p.98 / Chapter 3.2.4 --- Detection of the expression of Ang-2 isoforms in adult rat testis --- p.99 / Chapter 3.3 --- Expression of Ang-3 in the testis and testicular cell types --- p.103 / Chapter 3.3.1 --- Detection of Ang-3 expression in the testis and primary testicular cells by RT-PCR --- p.103 / Chapter 3.3.2 --- Detection of Ang-3 expression in testicular cell lines by RT-PCR --- p.105 / Chapter 3.3.3 --- Sequence analysis of Ang-3 transcripts amplified from TM4 mouse Sertoli cells and adult rat testis --- p.105 / Chapter 3.4 --- Expression of Tie 1 and Tie 2 in the testis and testicular blood vessel --- p.110 / Chapter 3.4.1 --- Detection of Tie 1 and Tie 2 expression in the testis and rat testicular blood vessel by RT-PCR --- p.110 / Chapter 3.4.2 --- Sequence analysis of Tie 1 transcripts amplified from adult rat testis and rat testicular blood vessel --- p.113 / Chapter 3.4.3 --- Sequence analysis of Tie 2 transcript amplified from rat testicular blood vessel --- p.113 / Chapter 3.5 --- "Western blot analysis of Ang-1 and Ang-2 expression in testicular tissues, primary testicular cells and cell lines" --- p.116 / Chapter 3.6 --- "Localization of Ang-1，Ang-2, Ang-4, Tie 1 and Tie2 proteins in adult rat testis by immunohistochemistry" --- p.122 / Chapter 3.7 --- "Comparison of angiopoietin expression patterns in testis using RT-PCR, Western immunoblotting and immunohistochemistry" --- p.128 / Chapter 3.8 --- Comparison of Tie 1 and Tie 2 expression patterns in testis using RT-PCR and immunohistochemistry --- p.128 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- "Expression of Ang-1 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.131 / Chapter 4.2 --- "Expression of Ang-2 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.136 / Chapter 4.3 --- "Expression of Ang-3 mRNA and protein in adult rat testis, mouse testis, rat testicular blood vessel, primary testicular cells and testicular cell lines" --- p.141 / Chapter 4.4 --- Expression of Tie 1 and Tie 2 mRNAs and proteinsin adult rat testis and rat testicular blood vessel --- p.143 / Chapter 4.5 --- Conclusion --- p.145 / Chapter 4.6 --- Future work --- p.146 / Chapter Chapter 5 --- References --- p.149

活性氧在內皮細胞生理發展比如細胞生長增殖和病理中起到非常重要的作用。在病理條件下，活性氧在血管功能失調和重構起到關鍵作用。氧化應激現在被認為存在於多種形式的心血管疾病中。諸多證據表明著活性氧誘導的心血管系統中很多功能異常之前會伴隨有細胞內鈣離子濃度的上升。 / 在本論文的第一個部分，我比較了活性氧在大血管（主動脈）和小血管（腸系膜動脈）的內皮細胞裡引起的鈣應激的相似和差異之處。在這兩種細胞中，活性氧均可引起細胞內鈣離子濃度的上升。這種鈣離子濃度增加可被磷酸酯酶C (PLC) 的抑製劑U73122或者磷酸肌醇受體 (IP₃R) 抑製劑 （Xestospongin C， XeC）大幅度的減弱。此外，用過氧化氫預處理後的細胞會降低細胞對ATP的鈣應激反應。這種鈣應激反應的抑制可能是由於過氧化氫引發的鈣庫流失。令人關注的是，腸系膜動脈的內皮細胞對過氧化氫的作用更為敏感。次黃嘌呤 (hypoxanthine; HX) 加上黃嘌呤(xanthine; XO) 也能引起這兩種內皮細胞鈣離子濃度的上升，而這種鈣離子的增加源於超氧陰離子而不是氫氧離子。在腸系膜動脈的內皮細胞中，過氧化氫在此事件中起到的作用明顯比在主動脈細胞大。總之，過氧化氫可以引起大血管和小血管的內皮細胞裡磷酸酯酶C-磷酸肌醇受體依賴的鈣應激反應。而這種鈣應激後的鈣庫耗竭會對ATP引起的鈣應激起作用。綜上所述，小血管的內皮細胞的鈣應激比大血管的內皮細胞對過氧化氫更為敏感。 / 基於以上的結果，在第二部分的內容中，我們以培植的微血管內皮細胞系（H5V）為小血管內皮細胞的模型，研究了TRPM2通道在過氧化氫誘導的的鈣應激和凋亡中的作用。TRPM2是表達在動物是血管內皮組織中的氧化敏感的和陽離子無選擇性通道。我們開發了TRPM2通道的抑制性抗體 (TM2E3)，這種抗體可以結合到TRPM2通道的離子孔道的E3區域。對H5V細胞進行TM2E3的預處理後，可以降低細胞對過氧化氫刺激下的鈣離子的增加。用TRPM2特異的短發卡核糖核酸 （shRNA）也有同樣的抑制反應。我們用了3種方法來檢測過氧化氫誘導的細胞凋亡：四甲基偶氮唑盐（MTT）檢測，脫氧核糖核酸凋亡片段的檢測和4,6-联脒-2-苯基吲哚(DAPI) 核染色。基於以上的試驗結果，TM2E3 和TRPM2特異的shRNA都表現出了對過氧化氫引起的細胞凋亡的保護作用。相反，在細胞中過表達TRPM2會導致過氧化氫引起的鈣離子濃度上升的增加和細胞凋亡程度的加重。 這些發現強有力的證明了TRPM2 介導了過氧化氫引起的鈣離子濃度的上升和細胞凋亡。此外，我們還研究了TRPM2激活後的下游事件：半胱氨酸蛋白酶-3，-8和9是否參與到這個過程。我的數據表明過氧化氫誘導細胞凋亡是通過內源和外源通路導致半胱氨酸酶-3激活，而TRPM2在這個過程中起到了重要的決定作用。總括而言，TRPM2 介導了過氧化氫誘導的內皮細胞凋亡，下調內源性的TRPM2的表達會保護血管內皮細胞。 / Reactive Oxygen Species (ROS) play a key role in normal physiological processes such as cell proliferation and growth, as well as in pathological processes. Under pathological conditions ROS contribute to vascular dysfunction and remodeling through oxidative damage. Oxidative stress is now thought to underlie many cardiovascular diseases. Accumulating evidence also demonstrate that many ROS-induced functional abnormalities in the cardiovascular system are preceded by an elevation of intracellular Ca²⁺. / In the first part, I compared the Ca²⁺ responses to ROS between mouse endothelial cells derived from large-sized artey aortas (aortic ECs), and small-sized mesenteric arteries (MAECs). Application of hydrogen peroxide (H₂O₂) caused an increase in cytosolic Ca²⁺ levels ([Ca²⁺]i) in both cell types. The [Ca²⁺]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP₃) receptors. In addition, treatment of endothelial cells with H₂O₂ reduced the Ca²⁺ responses to subsequent challenge of ATP. The decreased Ca²⁺ responses to ATP were resulted from a pre-depletion of intracellular Ca²⁺ stores by H₂O₂. Interestingly, we also found that Ca²⁺ store depletion was more sensitive to H₂O₂ treatment in endothelial cells derived from mesenteric arteries than those of derived from aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca²⁺]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H₂O₂ but not by hydroxyl radicals. H₂O₂ made a greater contribution to HX-XO-induced [Ca²⁺]i rises in endothelial cells from mesenteric arteries than those from aortas. In summary, H₂O₂ could induce store Ca²⁺ release via phospholipase C-IP₃ pathway in endothelial cells. Emptying of intracellular Ca²⁺ stores contributed to the reduced Ca²⁺ responses to subsequent ATP challenge. Furthermore, the Ca²⁺ responses in endothelial cells of small-sized arteries were more sensitive to H₂O₂ than those of large-sized arteries. / In the second part, I used murine heart microvessel endothelial cell line H5V as a model of endothelial cells from small-sized arteries to investigate the role of Melastatin-like transient receptor potential channel 2 (TRPM2) channels in H₂O₂-induced Ca²⁺ responses and apoptosis. TRPM2 is an oxidant-sensitive cationic non-selective channel that is expressed in mammalian vascular endothelium. A TRPM2 blocking antibody channel (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the Ca²⁺ responses to H₂O₂. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect in H₂O₂-induced Ca²⁺ responses. H₂O₂-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect on H₂O₂-induced apoptotic cell death. In contrast, overexpression of TRPM2 in H5V cells increased the Ca²⁺ responses to H₂O₂ and aggravated the apoptotic cell death in response to H₂O₂. These findings strongly suggest that the TRPM2 channel mediates Ca²⁺ overload in response to H₂O₂ and contributes to oxidant-induced apoptotic cell death in vascular endothelial cells. I also examined the downstream cascades of TRPM2 activation and explored whether caspase-3, -8 and -9 were involved in this process. My data indicates that H₂O₂-induced cell apoptosis through both intrinsic and extrinsic apoptotic pathways, leading to activation of caspases-3. Furthermore, TRPM2 played an essential role in the process. Together, my data suggest that TRPM2 mediates H₂O₂-induces endothelial cell death and that down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Lei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 101-114). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Declaration of Originality --- p.I / Abstract --- p.II / 論文摘要 --- p.IV / Acknowledgments --- p.VI / Abbreviations and Units --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Reactive oxygen species and Reactive nitrogen species --- p.1 / Chapter 1.1.1 --- What is oxidative stress? --- p.1 / Chapter 1.1.2 --- Types of ROS --- p.2 / Chapter 1.1.2.1 --- Hydroxyl radical (*OH) --- p.2 / Chapter 1.1.2.2 --- Hydrogen peroxide (H₂O₂) --- p.3 / Chapter 1.1.2.3 --- Superoxide (O₂*⁻) --- p.4 / Chapter 1.1.2.4 --- Nitric oxide (NO) --- p.5 / Chapter 1.1.3 --- ROS-producing systems --- p.6 / Chapter 1.1.3.1 --- NAD(P)H oxidase --- p.6 / Chapter 1.1.3.2 --- Xanthine oxidase (XO) --- p.7 / Chapter 1.1.3.3 --- The mitochondrial respiratory chain --- p.8 / Chapter 1.1.3.4 --- Uncoupled endothelial NO synthase --- p.8 / Chapter 1.1.4 --- Antioxidant defense mechanisms in the cardiovascular systems --- p.9 / Chapter 1.1.4.1 --- SOD --- p.9 / Chapter 1.1.4.2 --- Catalase --- p.10 / Chapter 1.1.4.3 --- Glutathione peroxidase (GPx) --- p.10 / Chapter 1.1.4.4 --- Small molecules --- p.11 / Chapter 1.1.5 --- Role of oxidative stress in human diseases --- p.12 / Chapter 1.1.6 --- Endothelium dysfunction in oxidative stress-relating human diseases --- p.12 / Chapter 1.1.7 --- Role of Ca²⁺ in oxidative stress-relating human diseases --- p.14 / Chapter 1.1.8 --- Differential effects of ROS on endothelial calcium signaling --- p.15 / Chapter 1.1.8.1 --- Multiple Oxidative Stress-induced Ca²⁺ signaling pathway --- p.16 / Chapter 1.1.9 --- Effects of ROS on Agonist-induced endothelial calcium signaling --- p.19 / Chapter 1.1.10 --- Role of H₂O₂ as EDHF --- p.20 / Chapter 1.1.11 --- Differential effect of ROS on cells derived from large-sized and small-sized artries --- p.21 / Chapter 1.2 --- The transient receptor potential (TRP) Channels --- p.21 / Chapter 1.2.1 --- TRP Channel structure --- p.22 / Chapter 1.2.2 --- TRP Channel function --- p.23 / Chapter 1.2.3 --- TRPM subfamily --- p.23 / Chapter 1.2.3.1 --- TRPM2 Property and Structure --- p.24 / Chapter 1.2.3.2 --- TRPM2 Expression --- p.25 / Chapter 1.2.3.3 --- TRPM2 Activator --- p.25 / Chapter 1.2.3.4 --- TRPM2 Physiological and pathophysiological function --- p.28 / Chapter Chapter 2 --- Objectives of the Present Study --- p.35 / Chapter Chapter 3 --- Materials and methods --- p.37 / Chapter 3.1 --- Ethics statement --- p.37 / Chapter 3.2 --- Materials --- p.37 / Chapter 3.3 --- Methods --- p.38 / Chapter 3.3.1 --- Cell culture --- p.38 / Chapter 3.3.1.1 --- Primary Cell Culture --- p.38 / Chapter 3.3.1.2 --- H5V endothelial cell line --- p.39 / Chapter 3.3.1.3 --- Human embryonic kidney 293 (HEK293) cells --- p.39 / Chapter 3.3.4. --- TRPM2-specific shRNA, TRPM2 and transfection --- p.39 / Chapter 3.3.5 --- Western blotting --- p.40 / Chapter 3.3.6 --- [Ca²⁺]i Studies --- p.43 / Chapter 3.3.6.1 --- Fluo-4/AM- Measuring intracellular [Ca²⁺]i --- p.43 / Chapter 3.3.6.2 --- Fura-2/AM-Measuring intracellular [Ca²⁺]i --- p.44 / Chapter 3.3.6.3 --- Mag-fluo-4-Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.45 / Chapter 3.3.7 --- IP₃ measurement --- p.45 / Chapter 3.3.8 --- Electrophysiology --- p.46 / Chapter 3.3.9 --- TRPM2 blocking antibody (TM2E3) and Pre-immune IgG Generation --- p.46 / Chapter 3.3.10 --- DNA fragmentation assay --- p.47 / Chapter 3.3.11 --- DAPI Staining --- p.48 / Chapter 3.3.12 --- MTT assay --- p.48 / Chapter 3.3.13 --- Statistical analysis --- p.49 / Chapter Chapter 4 --- Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca²⁺: Comparison of Endothelial Cells from Large-sized and Small-sized Arteries --- p.50 / Chapter 4.1 --- Introduction --- p.50 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.2.1 --- Primary Cell Culture --- p.52 / Chapter 4.2.2 --- [Ca²⁺]i Measurement --- p.52 / Chapter 4.2.3 --- Measuring Ca²⁺ Content in Intracellular Ca²⁺ Stores --- p.52 / Chapter 4.2.4 --- IP₃ measurement --- p.53 / Chapter 4.2.5 --- Data Analysis --- p.53 / Chapter 4.3 --- Results --- p.53 / Chapter 4.3.1 --- Both Ca²⁺ entry and store Ca²⁺ release contributed to H₂O₂-induced [Ca²⁺]i rises.. --- p.53 / Chapter 4.3.2 --- H₂O₂ enhanced IP₃ production and store Ca²⁺ release --- p.54 / Chapter 4.3.3 --- H₂O₂ reduced the Ca²⁺ responses to ATP in a H₂O₂ concentration and incubation time dependent manner --- p.54 / Chapter 4.3.4 --- H₂O₂ induced Ca²⁺ store depletion --- p.55 / Chapter 4.3.5 --- Ca²⁺ responses to ATP in the absence of H₂O₂ --- p.56 / Chapter 4.3.6 --- Non-involvement of hydroxyl radical --- p.56 / Chapter 4.3.7 --- HX-XO-induced [Ca²⁺]i rises were caused by superoxide anion and hydrogen peroxide --- p.56 / Chapter 4.4 --- Discussion --- p.68 / Chapter Chapter 5 --- Role of TRPM2 in H₂O₂-induced cell apoptosis in endothelial cells --- p.72 / Chapter 5.1 --- Introduction --- p.72 / Chapter 5.2 --- Materials and Methods --- p.73 / Chapter 5.2.1 --- Cell Culture --- p.74 / Chapter 5.2.2 --- [Ca²⁺]i measurement --- p.74 / Chapter 5.2.3 --- DNA fragmentation assay --- p.74 / Chapter 5.2.4 --- MTT assay --- p.74 / Chapter 5.2.5 --- TRPM2-specific shRNA, TRPM2 and transfection --- p.75 / Chapter 5.2.6 --- Electrophysiology --- p.75 / Chapter 5.2.7 --- Western blotting --- p.75 / Chapter 5.2.8 --- DAPI Staining --- p.76 / Chapter 5.2.9 --- Data analysis --- p.76 / Chapter 5.3 --- Results --- p.76 / Chapter 5.3.1 --- Involvement of TRPM2 channels in H₂O₂-induced Ca²⁺ influx in H5V cells --- p.76 / Chapter 5.3.2 --- Involvement of TRPM2 channels in H₂O₂-elicited whole-cell current change in H5V cells --- p.77 / Chapter 5.3.3 --- Role of TRPM2 channels in H₂O₂-induced apoptotic cell death in H5V cells --- p.78 / Chapter 5.3.4 --- Involvement of caspases in H₂O₂-induced apoptotic cell death --- p.79 / Chapter 5.3.5 --- Involvement of TRPM2 in TNF-α-induced cell death in H5V cells --- p.79 / Chapter 5.3 --- Discussion --- p.90 / Chapter Chapter 6 --- General Conclusions, Disscussion and Future work --- p.94 / Chapter 6.1 --- General Conclusions --- p.94 / Chapter 6.2 --- Discussion --- p.95 / Chapter 6.2.1. --- Comparative study --- p.95 / Chapter 6.2.2. --- IP₃ receptor (IP₃R) --- p.95 / Chapter 6.2.3. --- TM2E3-Specific blocking antibody of TRPM2 --- p.95 / Chapter 6.2.4. --- Pathological effect of H₂O₂ at high concentration --- p.96 / Chapter 6.2.5 --- Non-change on Basal [Ca²⁺]i --- p.97 / Chapter 6.3. --- Future work --- p.98 / References --- p.101

Vascular diseases, such as stroke and heart attacks, account for more than 50% of abnormal death worldwide. The cause of these diseases is linked to malfunctions of vascular endothelial cells, in particular the endothelial glycocalyx. This study investigates the location and stability of the endothelial glycocalyx under different flow conditions in vitro. AFM (Atomic Force Microscopy) micro indentation is carried out on endothelial cell membrane to determine its Young's modulus. The Young's modulus of the glycocalyx layer is then deduced from measurements on cell membranes with, and those without, the glycocalyx layer. Heparan sulphate (HS) is an important component of the glycocalyx and can be removed by the enzyme heparinase-III (Hep-III). Our results show the glycocalyx on cultured Human Umbilical Vein Endothelial Cells (HUVECs) has a Young's modulus of ~0.64Kpa. We further observe how the Young's modulus of the endothelial cell membrane decreases with time, as the glycocalyx layer redevelops, following its removal by Hep-III. Steady and oscillatory shear stimulations are used in flow chamber experiments. Under 24 hours' steady shear stimulation (12.6 dyn/cm2), cells are seen to elongate and reorient parallel to the flow direction. The glycocalyx is seen to shift to the peripheral region of the cell surface. With actin depolymerisation treatment, significant shedding of the glycocalyx from the luminal surface of the cell is observed. This occurs together with the loss of focal adhesions on the basal membrane. When endothelial cells are subjected to 24 hours' oscillating shear stress, the size of the cell increases as the oscillatory reversal time (time between changes in oscillatory flow direction) increases. Measurements are taken with oscillatory flow reversal programmed at 5s, 10s and 15s. The angle (between the long axis of the cell and the flow direction) and the aspect ratio (long axis vs short axis) change from 41.57° and 1.72 : 1 (static) to 40.18° and 3.26 : 1 (5s), 36.71° and 4.17 : 1 (10s), 26.5° and 4.39 : 1 (15s). Both the height and the area of the cell increase. The Young's modulus of the endothelial cell membrane is measured under oscillatory flows with different reversal time and compared to that under static flow conditions. An increase in the Young's modulus is observable under oscillatory flows, with the most significant change occurring at the edge (i.e. periphery) of the cell membrane area. As the oscillatory reversal time increases from 5s to 15s, the Young's modulus of the cell membrane increases. In the apical areas of the cell membrane, the increase is less significant. These results indicate that the thickness of the glycocalyx decreases as cells are exposed to oscillatory flows, and the loss is most significant in the peripheral region of the cell membrane. As the oscillatory reversal time increases from 5s to 15s, so the loss in the glycocalyx increases.