Vascular endothelial cells (VECs) maintain vascular integrity by regulating the passage of solutes, macromolecules, and cells between the vascular and perivascular space and are critical in a wide number of physiological processes, such as the delivery of nutrients and oxygen to surrounding tissues, leukocyte trafficking, angiogenesis, and tissue repair. VEC permeability is regulated, at least in part, by VE-cadherin-based adherens junctions that coordinate inter-VEC contacts and communicate the strength of these interactions to the cell via the actin cytoskeleton. Although the ubiquitous second messenger, cyclic adenosine 3'€™, 5'€™-monophosphate (cAMP), has been shown to reduce VEC permeability, the molecular basis of this effect is currently unclear. Herein, we report that cAMP and its two effectors, cAMP-dependent protein kinase A-II (PKA-II) and exchange protein activated by cAMP-1 (EPAC1), improve barrier function and differentially coordinate this effect through both VE-cadherin and actin cytoskeletal structures. We have also identified cyclic nucleotide phosphodiesterase (PDE) 4 as the major PDE regulating VEC barrier function. Through the use of cAMP-elevating agents and RNAi-mediated knockdown of PKA-Cα, EPAC1 and PDE4D, we have identified a dominant role for EPAC1 in VEC permeability as well as recognized PDE4D as a potential adaptor protein VE-cadherin-based complexes. Our results are consistent with previous reports of a role for both PKA and EPAC1 in controlling VE-cadherin mediated barrier function and additionally provide novel insight into the differential roles that PKA, EPAC1 and PDE4D play in stabilizing VEC barrier function. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-09-18 16:09:59.12

ANCA-associated vasculitis is a group of devastating autoimmune diseases that predominantly target and destroy small blood vessels. The interaction of neutrophils and monocytes with the endothelial cell lining of blood vessels is imperative to understanding the pathophysiology of the disease. The nature and temporal dynamics of these interactions are mostly unknown and could provide a currently unmet clinical need for more reliable biomarkers of disease activity. This study describes the development of a 4-dimensional in vitro live cell imaging model allowing the interactions of leukocytes with endothelial cells to be analysed in the context of health and disease. Monocytes and neutrophils were isolated from peripheral venous blood of AAV patients and healthy donors. Cells were fluorescently labelled and imaged on a monolayer of human umbilical vein endothelial cells (HUVEC) using a spinning disc confocal microscope. Leukocyte migration, partial and full transmigration, route of transmigration, degranulation and the presence of leukocyte-derived particles inside endothelial cells were measured and the influence of ANCA or BVAS status considered. Following a series of preliminary experiments, it was determined that neutrophil degranulation and partial transmigration indicated early promise as potential biomarkers of disease activity. Several circulating serum analytes correlated with in vitro leukocyte functions, complementing these findings but also highlighting the prevalent immune dysfunction in the pathophysiology and development of the disease. Fatigue is a common symptom within the AAV community and the complex relationship between autoimmune fatigue and leukocyte functions was examined. The data in this thesis describes the development of a novel in vitro live cell imaging platform which can be used to investigate leukocyte functions as potential markers of disease activity as well as understanding their role in the pathophysiology of AAV.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2008. / Härtill 3 uppsatser.

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.

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.

Many studies have demonstrated that there is a strong relationship between endothelial dysfunction and oxidative stress and have demonstrated also that hyperglycemia is associated with increased generation of oxidative stress and atherosclerotic vascular diseases, but we do not know how hydrogen peroxide (H2O2) and high glucose (HG) could affect calcium regulatory proteins of human vascular endothelial cells (HUVECs) in vitro. In the present study, we have examined the acute effect of H2O2 (100 M) and the effect of chronic exposure to HG concentration (35 mM) on the calcium regulatory system of human vascular endothelial cells using fluorescence imaging microscopy (fura-2). In this study, we tested the hypothesis that calcium regulatory proteins (SERCA-ATPase and PMCA-ATPase pumps and the NCX exchanger) of ECs have different sensitivities to H2O2 and high glucose concentration. We also tested the hypothesis that calcium regulatory proteins could be potential targets of ROS at the early stage of vascular disease. The results of this study showed that both H2O2 and high glucose induced significant delay in calcium removal time (CRT). The study of H2O2 showed that the delay in CRT was due to partial inhibition of SERCA-ATPase and the sodium calcium exchanger (NCX) activity and the effect of H2O2 on CRT was reversible. In contrast, the PMCA-ATPase pump was resistant to inhibition by H2O2. Furthermore, H2O2 induced a 40 ± 6.5 % reduction in endoplasmic reticulum refilling. The second part of the study showed that exposure of ECs to HG concentration for 10 days induced a significant delay in CRT and this delay was due to partial blockade of the SERCA-ATPase pump. Blockade of PMCA-ATPase pump with vanadate showed a further delay in CRT. We conclude that: 1- Both H2O2 and HG affected components of the calcium removal system with different sensitivities; 2- H2O2 and HG did not show any inhibitory effects on the PMCA-ATPase pump; 3- The effect of H2O2 on CRT was reversible; 4- The effect of HG on CRT could be due to increased production of H2O2; 5- The calcium regulatory proteins of ECs could be potential targets for ROS during the early stage of a cardio-vascular disease such as diabetes mellitus.

To understand whether integrin β4 is involved in vascular endothelial cell (VEC) senescence, we examined integrin β4 level changes, as well as P53 and reactive oxygen species (ROS) levels and alterations of phosphatidylcholine-specific phospholipase C (PC-PLC) activity before and after knocking-down integrin β4 by small interfering RNA. We found integrin β4, P53 and ROS levels increased significantly, while Ca2+-independent PC-PLC activity obviously decreased during VEC senescence. On the other hand, integrin β4 down-regulation attenuated the senescence phenotype and reversed Ca2+-independent PC-PLC activity, and P53 and ROS levels. The data suggested that integrin β4 might mediate VEC senescence through depressing Ca2+-independent PC-PLC and elevating the levels of P53 and ROS.

At present, vascular endothelial cell (VEC) apoptosis induced by deprivation of fibroblast growth factor-2 (FGF-2) and serum has been well studied. But how to trigger VEC apoptosis in the presence of FGF-2 and serum is not well known. To address this question, in this study, the effects of safrole oxide on angiogenesis and VEC growth stimulated by FGF-2 were investigated. The results showed that safrole oxide inhibited angiogenesis and induced VEC apoptosis in the presence of FGF-2 and serum. To understand the possible mechanism of safrole oxide acting, we first examined the phosphorylation of Akt and the activity of nitric oxide synthase (NOS); secondly, we analyzed the expressions and distributions of Fas and P53; then we measured the activity of phosphatidylcholine specific phospholipase C (PC-PLC) in the VECs treated with and without safrole oxide. The results showed that this small molecule obviously suppressed Akt phosphorylation and the activity of NOS, and promoted the expressions of Fas and P53 markedly. Simultaneously, Fas protein clumped on cell membrane, instead of homogenously distributed. The activity of PC-PLC was not changed obviously. The data suggested that safrole oxide effectively inhibited angiogenesis and triggered VEC apoptosis in the presence of FGF-2 and serum, and it might perform its functions by suppressing Akt/NOS signal pathway, upregulating the expressions of Fas and P53 and modifying the distributing pattern of Fas in VEC. This finding provided a powerful chemical probe for promoting VEC apoptosis during angiogenesis stimulated by FGF-2.

Atherosclerotic cardiovascular disease is a leading cause of death in the United States and worldwide. While much progress has been made in investigating dyslipidemia and inflammation regarding atherosclerotic disease, much is still unknown about the role of endogenous vascular cells in atherosclerosis. More importantly, as targeting dyslipidemia and inflammation has yielded successful therapies, can therapeutically targeting vascular dysfunction enhance existing therapies for treating cardiovascular disease? In this thesis, I sought to investigate the role of the matrix metalloproteinase, ADAMTS7, a gene implicated in atherosclerosis by genome-wide association studies (GWAS). Subsequent to the human genetic studies associating ADAMTS7 with atherosclerotic cardiovascular disease, in vivo investigations demonstrated that ADAMTS7 is proatherogenic and induced in response to vascular injury. However, the mechanisms governing ADAMTS7's function and the causal cell type responsible for producing ADAMTS7 remain unclear. To determine where ADAMTS7 expression occurs in atherosclerosis, we interrogated the largest single-cell RNA sequencing dataset of human carotid atherosclerosis. We found ADAMTS7 expression in endothelial cells, smooth muscle cells (SMCs), fibroblasts, and mast cells. We subsequently created both endothelial and SMC-specific Adamts7 conditional knockout and transgenic mice. The conditional knockout of Adamts7 in either cell type is insufficient to reduce atherosclerosis, but transgenic induction in either cell type increases peripheral atherosclerosis. In SMC transgenic mice, this increase coincides with decreased plaque stability and an expansion of lipid-laden SMC foam cells. RNA sequencing in SMCs revealed an upregulation of lipid uptake genes typically assigned to macrophages. Subsequent experiments demonstrated that Adamts7 increases SMC oxLDL uptake through Cd36. Furthermore, Cd36 expression is increased due to an Adamts7-mediated increase in Spi1, a known myeloid cell fate transcription factor. In summary, Adamts7 is expressed by multiple vascular cell types during atherosclerosis, and in SMCs, Adamts7 promotes oxLDL uptake, thereby increasing SMC foam cell and atherosclerosis. While investigating ADAMTS7, we sought to identify a cell surface persistent marker of SMCs to aid investigations into ADAMTS7. SMCs play a central role in the development of atherosclerosis due in part to their capability to phenotypically transition into either a protective or harmful state. However, the ability to identify and trace SMCs and their progeny in vivo is limited due to the lack of well-defined SMC cell surface markers. Therefore, investigations into SMC fate must utilize lineage-tracing mouse models, which are time-consuming and challenging to generate and not feasible in humans. We, thus, employed CITE-seq to phenotypically characterize the expression of 119 cell surface proteins in mouse atherosclerosis. We found that CD200 is a highly expressed and specific marker of SMCs, which persists even with phenotypic modulation. We validated our findings using a combination of flow cytometry, qPCR, and immunohistochemistry, all confirming that CD200 can identify and mark SMCs and their derived cells in early to advanced mouse atherosclerotic lesions. Additionally, we describe a similar expression pattern of CD200 in human coronary and carotid atherosclerosis. Thus, CD200 is a lineage marker for SMCs and SMC-derived cells in mouse and human atherosclerosis. In conclusion, this body of work investigated the role of vascular cells in atherosclerosis. We have identified a new marker of SMCs, adding an additional tool that can be broadly employed to investigate the vasculature. In addition, we have mechanistically unraveled how one vascular GWAS hit, ADAMTS7, can perpetuate atherosclerosis. Our findings demonstrate that ADAMTS7 can promote foam cell expansion in atherosclerosis. While more work is needed to understand the role of these SMC foam cells in atherosclerosis, our investigations thus far have demonstrated that ADAMTS7 can greatly expand these cells. As such, our work supports the development of a drug to inhibit ADAMTS7 for treating atherosclerotic cardiovascular disease.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Exposure to harmful stimuli can render vascular endothelial cells dysfunctional, characterised by reduced nitric oxide (NO) bioavailibility. Endothelial dysfunction (ED) is a reversible precursor of ischaemic heart disease (IHD), and understanding the mechanisms underlying the development of ED could lead to clinical strategies in preventing/treating IHD. Very little is known about the responses of cardiac microvascular endothelial cells (CMECs) to pro-ED stimuli, as most studies are conducted on macrovascular endothelial cells. The current dissertation set out to comprehensively investigate the responses of cultured primary adult rat CMECs to known harmful stimuli, viz. hypoxia and tumor necrosis factor-alpha (TNF-α; proinflammatory cytokine). We were interested to investigate whether this distinct endothelial cell type would develop classical features of ED, and if so, what the underlying mechanisms were. First we aimed to establish a baseline characterization of the CMECs under control conditions. Next, we developed a model of hypoxia-induced cell injury and measured apoptosis/necrosis, intracellular NO and reactive oxygen species (ROS), expression and activation of signalling proteins involved with NObiosynthesis, hypoxia and apoptosis, and differential regulation of proteins. Finally, we characterised CMEC responses to treatment with TNF-α. We assessed apoptosis/necrosis, intracellular NO and ROS levels, NO-biosynthesis pathway proteins and large-scale differential protein regulation. The above measurements were performed by morphological assessment (light and fluorescence microscopy), FACS analysis, western blotting and large-scale proteomic analyses. Data showed that CMECs shared many baseline features with other endothelial cell types, including morphological appearance, LDL-uptake, NO-production, and expression of eNOS protein. In a novel observation, proteomic analysis revealed the expression of 1387 proteins. Another novel finding was the high abundance of structural mitochondrial proteins, suggesting that CMECs require mitochondria for non-respiration purposes as well. High expression of vesicle, glycolytic and RAS signalling proteins were other features of the baseline CMECs. CMECs exposed to hypoxia responded by increased apoptosis/necrosis and expression of the hypoxia-marker, HIF-1α. Interestingly, hypoxic CMECs showed increased eNOS-NO biosynthesis, associated with increased mitochondrial ROS and reduced anti-oxidant systems, suggestive of oxidative stress. In accordance with the literature, several glycolytic proteins were up-regulated. A novel finding was the up-regulation of proteins involved with protein synthesis, not usually described in hypoxic cell studies. The CMECs responded to TNF-α-treatment by exhibiting hallmarks of ED, namely attenuated biosynthesis of PKB/Akt-eNOSderived NO and the development of outspoken response to oxidative stress as indicated by the up-regulation of several anti-oxidant systems. The data showed that TNF-α treatment elicited classical TNF-Receptor 1-mediated signalling characterized by the dual activation of pro-apoptotic pathways (BID and caspase-3) as well as the protective, pro-inflammatory IKB-alpha–NF-KB pathway. In conclusion, this is the first study of its kind to describe a comprehensive characterisation of CMECs under baseline and injury-inducing conditions. On the whole, although it appeared as if the CMECs shared many responses and mechanisms with more frequently researched endothelial cell types, the data also supplied several novel additions to the literature, particularly with the application of proteomics. We believe that this dissertation has provided more insights into endothelial heterogeneity in the vascular system and into the mechanisms adopted by CMECs when exposed to stimuli typically associated with cardiovascular risk. / AFRIKAANSE OPSOMMING: Blootstelling aan skadelike stimuli kan tot disfunksionaliteit van vaskulêre endoteelselle lei wat deur verlaagde biobeskikbaarheid van stikstofoksied (NO) gekenmerk word. Endoteeldisfunksie (ED) is ‘n omkeerbare voorganger van isgemiese hartsiekte (IHD), en ‘n beter begrip van die onderliggende meganismes van ED kan lei tot die ontwikkeling van kliniese strategieë vir die voorkoming/behandeling van IHD. Baie min is bekend oor die respons wat in kardiale mikrovaskulêre endoteelselle (CMECs) uitgelok word na blootstelling aan pro-ED stimuli, omdat meeste studies op makrovaskulêre endoteelselle uitgevoer word. Die huidige proefskrif het daarna gemik om die respons van primêre kulture van volwasse rot CMECs op bekende skadelike stimuli, nl. hipoksie en tumor nekrose faktor-alfa (TNF-α; pro-inflammatoriese sitokien) in diepte te ondersoek. Ons was veral geïnteresseerd om vas te stel of hierdie spesifieke endoteelseltipe die klassieke kenmerke van ED sou ontwikkel, en indien wel, wat die onderliggende meganismes sou wees. Eerstens het ons beoog om ‘n basislyn karaterisering van CMECs onder kontrole toestande daar te stel. Vervolgens het ons ‘n model van hipoksie-geïnduseerde selskade gevestig en apoptose/nekrose, intrasellulêre NO en reaktiewe suurstofspesies (ROS), sowel as die uitdrukking en aktivering van proteine betrokke by NO-biosintese, hipoksie en apoptose en differensiële regulering van proteine gemeet. Laastens het ons die respons van CMECs op behandeling met TNF-α gekarakteriseer. Ons het apoptose/nekrose, intrasellulêre NO en ROS vlakke, NO-biosintese-seintransduksieproteïene en grootskaalse differensiele regulering van proteïene gemeet. Bg. metings is uitgevoer deur gebruik te maak van morfologiese evaluasie (lig -en fluoressensiemikroskopie), vloeisitometriese analises, western blot analises en proteomiese analises. Data het getoon dat die basislyn eienskappe van CMECs grootliks met dié van ander endoteelseltipes ooreenstem, insluitende morfologiese voorkoms, LDL-opname, NO-produksie en die uitdrukking van eNOS proteïen. In ‘n nuwe waarneming, het die proteomiese data die uitdrukking van 1387 proteïene aangetoon. ‘n Ander nuwe bevinding was die voorkoms van ‘n groot aantal strukturele mitokondriale proteïene, wat daarop dui dat die CMECs mitokondria ook vir nie-respiratoriese doeleindes gebruik. ‘n Hoë uitdrukking van vesikulêre, glikolitiese en RAS-seintransduksie proteïene was ook kenmerkend van die basislyn CMECs. CMECS wat aan hipoksie blootgestel is, het reageer met ‘n verhoging in apoptose / nekrose en verhoogde uitdrukking van die hipoksie merker, HIF-1α. ‘n Interressante bevinding was dat eNOS-NO biosintese sterk toegeneem het in die hipoksiese CMECs wat met verhoogde mitokondriale ROS en verlaagde anti-oksidant sisteme (aanduidend van oksidatiewe stres) gepaardgegaan het. In ooreenstemming met die literatuur, is verskeie glikolitiese proteïene opgereguleer. ‘n Nuwe waarneming was die opregulering van proteïene wat betrokke is by proteïensintese, iets wat nie normaalweg in hipoksie-studies beskryf word nie. Die CMECs het op TNF-α behandeling gerespondeer deur tekens van ED te toon, naamlik ‘n afname in die NO afkomstig van PKB/Akt-eNOS biosintese en die ontwikkeling van uitgesproke reaksie op oksidatiewe stres soos aangedui deur die opregulering van verskeie anti-oksidant sisteme. Die data het ook aangedui dat TNF-α behandeling tot klassieke TNF-reseptor 1 bemiddelde seintransduksie gelei het, wat gekenmerk was deur die tweeledige aktivering van pro-apoptotiese seintransduksiepaaie (BID en kaspase-3) sowel as die beskermende, pro-inflammatoriese IKB-alpha-NF-KB seintransduksiepad. Ten slotte: hierdie is die eerste studie van sy soort wat die kenmerke en response van CMECs onder basislyn en pro-besering omstandighede in diepte beskryf. Alhoewel dit oor die algemeen wil voorkom asof die CMECs baie in gemeen het met ander, beter nagevorste endoteelseltipes, het die data egter ook verskeie nuwe bevindinge tot die bestaande literatuur gevoeg, spesifiek die data afkomstig van die proteomiese analises. Ons glo dat hierdie proefskrif meer insig verleen t.o.v. die heterogeniteit van vaskulêre endoteelselle asook t.o.v. die megansimes wat deur CMECs aangewend word wanneer hulle aan skadelike stimuli (geassosieer met kardiovaskulêre risiko) blootgestel word.