Extracellular Ub is an immune modulator that plays a role in suppression of inflammation, organ injury, myocyte apoptosis, and fibrosis. The purpose of this study was to investigate the effects of extracellular Ub on the process of cardiac angiogenesis. CMECs and aortic tissue were isolated from rats to measure changes in angiogenic protein levels and to assess angiogenic responses to extracellular Ub. In CMECs, extracellular Ub increased protein levels of VEGF-A and MMP-2, known angiogenesis regulators. CMECs demonstrated enhanced rearrangement of fibrillar actin and migration in response to Ub treatment. Ub-treated CMECs demonstrated an increase in tube network formation which was inhibited by the CXCR4 receptor antagonist, AMD3100. Methylated Ub, unable to form polyubiquitin chains, enhanced tube network formation. Aortic ring sprouting assays demonstrated that Ub increases microvessel sprouting in the Matrigel. The results of our study suggest a novel role for extracellular Ub in cardiac angiogenesis, providing evidence that extracellular Ub, at least in part acting via the CXCR4 receptor, has the potential to facilitate the process of angiogenesis in myocardial endothelial cells.

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.

Thesis (PhD)-- Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: The cardioprotective actions of the hormone melatonin against myocardial ischaemiareperfusion injury (IRI) are well-established. It has recently been shown to prevent the harmful effects of hyperphagia-induced obesity on the susceptibility of the heart to IRI as well as many of the harmful effects of obesity and insulin resistance. However, the exact mechanism whereby it exerts its beneficial action is still unknown. The aims of this study were to determine the effects of relatively short-term melatonin treatment in a rat model of diet-induced obesity on: (i) biometric and metabolic parameters, lipid peroxidation, myocardial IRI and intracellular signalling (ii) mitochondrial oxidative phosphorylation function (iii) cardiomyocyte glucose uptake and intracellular signalling. In addition, the effects of acute melatonin treatment of cardiac microvascular endothelial cells (CMEC) were determined on cell viability, nitric oxide production (NO), TNF- -induced dysfunction and intracellular signalling. Material and Methods: Male Wistar rats were randomly allocated to two groups for 20 weeks feeding with either standard rat chow or a high calorie diet. Each group was subdivided into 3 groups receiving either water throughout or melatonin (4mg/kg/day, in the drinking water) for the last 6 or 3 weeks of the experimental programme. Hearts, perfused in the working mode, were subjected to ischaemia/reperfusion and infarct size determined. Mitochondria and cardiomyocytes were isolated according to standard techniques and oxidative function and glucose uptake respectively determined. CMEC NO production and cell viability were quantified by FACS analysis of the fluorescent probes, DAF-2/DA and propidium iodide/Annexin V respectively. Intracellular signalling was evaluated using Western blot and appropriate antibodies. Results: The high-calorie diet caused significant increases in body weight gain, visceral adiposity, fasting blood glucose, serum insulin, triglycerides, HOMA-IR index and a concomitant reduction in serum adiponectin levels as well as larger myocardial infarct sizes after exposure to IRI compared to the control, indicating increased susceptibility to damage. Three as well as six weeks of melatonin administration to obese and insulin resistant rats reduced serum insulin levels and the HOMA-IR index. Myocardial infarct size was reduced in both control and diet groups. These effects were associated with increased activation of baseline myocardial STAT- 3 and the RISK pathway during reperfusion. The diet had no effect on the oxidative phosphorylation capacity of mitochondria, isolated from non-perfused hearts (baseline), but melatonin administration for 6 weeks induced a reduction in state 3 respiration rate; mitochondria isolated from diet hearts subjected to global ischaemia, exhibited an attenuated oxidative phosphorylation process which was improved by melatonin treatment. Melatonin in vitro enhanced cardiomycyte insulin stimulated glucose uptake of normal young rats but not of insulin resistant rats. In vivo melatonin treatment for 6 weeks increased basal (in diet group) and insulin stimulated glucose uptake in both control and diet groups. Melatonin (1nM) in vitro caused a significant reduction in necrosis and apoptosis of cultured CMEC, associated with a decrease in nitric oxide availability and eNOS activation and a concomitant increase in PKB/Akt, p38MAPK and AMPK activation. The harmful effects of TNF- treatment on signalling in CMEC could be prevented by co-treatment with melatonin. Conclusions: The results suggest that short-term melatonin treatment was able to significantly attenuate the diet-induced increased myocardial susceptibility to ischaemia/reperfusion damage. It may also improve cardiac glucose homeostasis and mitochondrial oxidative phosphorylation in an insulin resistant state. Melatonin in vitro protects CMEC against apoptosis and necrosis and reduces nitric oxide availability. These beneficial effects of melatonin may ultimately be due to its antioxidant capacity or receptor-mediated actions, but this remains to be established. / AFRIKAANSE OPSOMMING: Inleiding: Die vermoë van die hormoon, melatonien, om die hart teen iskemie/ herperfusiebesering (IHB) te beskerm, is welbekend. Onlangs is ook getoon dat melatonien IHB en verskeie van die nadelige effekte van vetsug en insulienweerstandigheid in hiperfagiegeïnduseerde vetsug kan voorkom. Die meganisme(s) betrokke by hierdie voordelige prosesse is egter grootliks onbekend. Die doel van hierdie studie was om die gevolge van korttermyn melatonienbehandeling in ‘n model van hiperfagiegeïnduseerde vetsug te ondersoek op (i) biometriese en metaboliese parameters, lipiedperoksidasie, miokardiale IHB en intrasellulêre seintransduksie, (ii) mitochondriale oksidatiewe fosforilasie, (iii) glukoseopname en intrasellulêre seintransduksie in kardiomiosiete en aanvullend, (iv) die invloed van akute melatonienbehandeling van kardiale mikrovaskulêre endoteelselle op sellulêre oorlewing, stikstofoksiedproduksie, TNF- - geïnduseerde disfunksie en seintransduksie. Metodiek: Manlike Wistarrotte is ewekansig in twee groep verdeel en vir 20 weke met standaard-rotkos of ‘n hoëkaloriedieet gevoer. Elke groep is in 3 subgroepe verdeel, wat deurgaans water of melatonien (4mg/kg/dag in die drinkwater) vir 3 of 6 weke voor die beëindiging van die eksperiment ontvang het. Harte is geperfuseer volgens die werkharttegniek, blootgestel aan iskemie/herperfusie en die infarktgrootte bepaal. Mitochondria en kardiomiosiete is volgens standaardtegnieke geïsoleer vir bepaling van oksidatiewe funksie en glukoseopname respektiewelik. NO produksie en sellewensvatbaarheid was gekwantifiseer deur vloeisitometriese analises (FACS) van die fluoresserende agense, DAF-2/DA en propidium jodied/Annexin V onderskeidelik. Intrasellulêre seintransduksie is evalueer met behulp van die Western kladtegniek en geskikte antiliggame. Resultate: Die hoëkaloriedieet het ‘n beduidende toename in liggaamsgewig, visserale vet, vastende bloedglukose, seruminsulienvlakke, trigliseriede, HOMA-IR-indeks en ‘n gepaardgaande verlaging in serumadiponektienvlakke tot gevolg gehad, sowel as groter miokardiale infarkte na iskemie/herperfusie. Laasgenoemde dui op ‘n groter vatbaarheid vir iskemiese beskadiging in harte van vetsugtige diere. Drie sowel as ses weke van melatonienbehandeling het die seruminsulienvlakke en HOMAindeks in vetsugtige diere beduidend verlaag, vergeleke met die kontroles. Miokardiale infarktgroottes was verminder in beide kontrole- en vetsuggroepe. Hierdie effekte het met ‘n verhoogde aktivering van basislyn STAT-3 en PKB/Akt en ERKp44/p42 tydens herperfusie gepaard gegaan. Die dieet het geen invloed op die oksidatiewe fosforilasiekapasiteit van mitochondria, geïsoleer uit harte van ongeperfuseerde harte, gehad nie (basislyn), maar melatonienbehandeling vir 6 weke het Staat 3 respirasie verlaag. Mitochondria, geïsoleer uit harte van vetsugtige rotte wat aan globale iskemie onderwerp was, het ‘n onderdrukte oksidatiewe fosforilasieproses gehad, wat egter deur melatonienbehandeling verbeter is. Melatonien in vitro het insuliengestimuleerde glukoseopname deur kardiomiosiete van jong, maar nie vetsugtige rotte nie, verhoog. In vivo melatonientoediening vir 6 weke het egter basale (in die dieetgroep) en insuliengestimuleerde glukoseopname in beide kontrole- en vetsuggroepe verhoog. Toediening van melatonien in vitro aan mikrovaskulêre endoteelselkulture het ‘n beduidende afname in nekrose, apoptose, stikstofoksied- beskikbaarheid en eNOS aktivering teweeggebring, tesame met ‘n verhoogde aktivering van PKB/Akt, p38MAPK en AMPK. Die nadelige effekte van TNF- toediening op seintransduksie in die mikrovaskulêre endoteelselle is deur melatonien voorkom. Gevogtrekkings: Die resultate toon dat melatonien ‘n merkwaardige beskermende effek op die toename in vatbaarheid vir iskemiese beskadiging in vetsugtige rotte gehad het. Dit mag ook miokardiale glukose-homeostase en mitochondriale oksidatiewe funksie in insulienweerstandigheid verbeter. Melatonien in vitro beskerm mikrovaskulêre endoteelselle teen nekrose asook apoptose en verminder die beskikbaarheid van stikstofoksied. Hierdie voordelige effekte van melatonien mag aan sy anti-oksidantvermoëns of stimulasie van die melatonienreseptor toegeskryf word, maar bewyse daarvoor ontbreek nog. / Division of Medical Physiology (Stellenbosch University), / National Research Foundation / Harry Crossley Foundation