ESE-1 is a novel transcriptional mediator of angiopoietin-1 expression in the setting of inflammation

Angiogenesis is a critical component of the inflammatory response associated with a number of conditions. Angiopoietin-1 (Ang-1) is an angiogenic growth factor that promotes the chemotaxis of endothelial cells and facilitates the maturation of new blood vessels. Ang-1 expression is up-regulated in response to tumor necrosis factor-alpha (TNF-alpha). To begin to elucidate the underlying molecular mechanisms by which Ang-1 gene expression is regulated during inflammation, we isolated 3.2 kb of the Ang-1 promoter that contain regulatory elements sufficient to mediate induction of the promoter in response to TNF-alpha, interleukin-1beta, and endotoxin. Surprisingly, sequence analysis of this promoter failed to reveal binding sites for transcription factors that are frequently associated with mediating inflammatory responses, such as NF-kappaB, STAT, NFAT, or C/EBP. However, putative binding sites for ETS and AP-1 transcription factor family members were identified. Interestingly, among a panel of ETS factors tested in a transient transfection assay, only the ETS factor ESE-1 was capable of transactivating the Ang-1 promoter. ESE-1 binds to specific ETS sites within the Ang-1 promoter that are functionally important for transactivation by ESE-1. ESE-1 and Ang-1 are induced in synovial fibroblasts in response to inflammatory cytokines, with ESE-1 induction slightly preceding that of Ang-1. Mutation of a high-affinity ESE-1 binding site leads to a marked reduction in Ang-1 transactivation by ESE-1, inducibility by inflammatory cytokines, and DNA binding to the ESE-1 protein. Transcriptional profiling of cells transiently transfected with an ESE-1 expression vector demonstrates that the endogenous Ang-1 gene is directly inducible by ESE-1. Finally, Ang-1 and ESE-1 exhibit a similar and strong expression pattern in the synovium of patients with rheumatoid arthritis. Our results support a novel paradigm for the ETS factor ESE-1 as a transcriptional mediator of angiogenesis in the setting of inflammation

Effects of Hydrogen Peroxide on Different Models of Wound Healing

Ph.DDOCTOR OF PHILOSOPH

Innovative biomedical applications using hybrid nanoparticles

Nanoparticle and metal-chelate technologies can be combined to create innovative hybrid materials with versatile functions for research and clinical purposes. This dissertation addresses the novel use of hybrid inorganic/organic nanoparticles in biomedical applications, including early detection of autoimmune disease and cancer as well as purification of therapeutic proteins. In an in vivo animal model of rheumatoid arthritis (RA), fluorescent nanoparticles coated with a magnetic resonance imaging (MRI) contrast enhancing metalchelate are used for cell specific trafficking of monocytes, which correlate with disease activity. Metal-chelate derivatized magnetic nanoparticles isolate tagged proteins, potential cancer therapeutics, from impurities generated from bacterial and mammalian cell expression systems. The novel applications of hybrid magnetic nanoparticles pursued in this dissertation have the ability to translate into new and more sensitive strategies to treat patients with chronic disease

Specific Levels of Therapeutic Ultrasound Stimulate the Release of Inflammatory and Angiogenic Mediators From Macrophages In Culture

Therapeutic ultrasound (TUS) is a treatment modality that is used to accelerate tissue healing. TUS is thought to affect cellular processes of tissue healing, especially those that occur in the inflammatory and early proliferative phases. TUS can be applied using various parameter selections including intensity, wavelength, duty cycle and treatment duration and no clear consensus exists on optimal parameters for healing enhancement. Macrophages are important mediators of inflammation and their actions are critical to normal progression into the proliferative phase of healing. They complete many functions during these periods of tissue healing, among those being release of cytokines and growth factors. These paracrine factors affect other inflammatory cells, resident cells of the healing tissue, including fibroblasts and endothelial cells that are necessary for restoration of damaged tissue. The hypothesis of this investigation is that TUS enhances early healing, in part, through stimulation of macrophage release of paracrine factors involved in coordination of the cellular aspects of tissue healing and that specific levels of TUS are most stimulatory for macrophages. This study examined macrophage release of interleukin-1beta (IL-1Beta), vascular endothelial growth factor (VEGF), transforming growth factor-Beta 1 (TGF-B1) and fibroblast mitogens, in response to varied levels of TUS. Fibroblasts incubated up to 48-hours in media conditioned by TUS-stimulated macrophages were not induced to proliferate regardless of the parameters sets of TUS applied. TUS (1 MHz, 400mW/cm2 SATA, 20% duty cycle, 10-minute exposure) induced macrophage release of VEGF and IL-1Beta within 10-minutes post-TUS, without any additional release being stimulated at 1-hour post-insonation. No other combination of TUS parameters studied induced release of IL-1Beta and VEGF. TUS did not induce release of TGF-Beta 1 at either time point post-TUS. VEGF and IL-1Beta release occurred in conjunction with lactate dehydrogenase (LDH) release from treated macrophages, indicating non-specific cell membrane permeabilization was involved in the cellular response. For IL-1Beta, TUS-stimulated release was inhibited at lower exposure temperatures. Inhibition of TUS-induced release at lower temperatures indicates that a cellular metabolic process, most likely exocytosis, was also stimulated by TUS. Based on these results, it appears that TUS exposure at 1 MHz, 400mW/cm2 SATA, 20% duty cycle induces non-specific and cell-mediated release of secretory proteins. Thus, enhanced release of cytokines and growth factors from macrophages is a possible mechanism by which TUS enhances tissue healing

Impact of circulating vascular endothelial growth factor level and the angiogenesis degree of the aspirated coronary thrombi on angiographic and clinical outcome of patients with acute ST elevation myocardial infarction treated with primary percutaneous coronary intervention

Koronarni tromb dobijen manuelnom aspiracionom trombektomijom od pacijenata sa akutnim infarktom miokarda sa elevacijom ST-segmenta (STEMI) lečenih primarnom perkutanom koronarnom intervencijom (pPCI) predstavlja pogodan materijal za proučavanje patofizioloških mehanizama u osnovi koronarne tromboze, trajanja ishemije kod STEMI pacijenata, kao i različitog obima angiogeneze u trombu. Vaskularni endotelni faktor rasta (VEGF) je ključni molekul angiogeneze, koji se može meriti u krvi STEMI pacijenata. Vrednosti cirkulišućeg VEGF-a mogu reflektovati kompenzatorni angiogeni odgovor ishemijskog miokarda i predstavljati važan prognostički faktor kod pacijenata sa infarktom miokarda. Cilj. Cilj ove studije bio je da se odredi starost i stepen angiogeneze aspiriranog koronarnog tromba i izmeri vrednost cirkulišućeg serumskog VEGF-a (sVEGF) u populaciji STEMI pacijenata lečenih pPCI uz aspiracionu trombektomiju, kao i ispitivanje njihovog uticaja na angiografske i kliničke ishode STEMI pacijenata. Materijal i metode. U istraživanje je uključeno 100 konsekutivnih STEMI pacijenata lečenih pPCI i aspiracionom trombektomijom od kojih je dobijena adekvatna količina materijala za dalju patohistološku analizu (>1 mm3). Aspirirani materijal analiziran je svetlosnom i konfokalnom mikroskopijom. Kategorizacija trombova prema starosti (svež, litički i organizovani tromb) izvršena je bojenjem hematoksilin-eozinom. Stepen angiogeneze je određen imunohistohemijskim bojenjem na markere endotelnih ćelija (EĆ) – CD34 i CD31, i gradiran kao pojedinačne EĆ, grupisane EĆ i kapilarne petlje EĆ. Vrednosti sVEGF-a prilikom prijema pacijenata u salu za kateterizaciju pre PCI određivane su ELISA metodom. Angiografski ishodi su podrazumevali analizu stepena miokardnog ispiranja (MBG) na finalnom angiogramu i stepena rezolucije ST-elevacije (STR) na elektrokardiogramu nakon 60 minuta od pPCI. Klinički ishodi procenjeni su veličinom infarkta miokarda na osnovu maksimalnih vrednosti oslobođenih kardiospecifičnih enzima tokom hospitalizacije (CKmax, troponin Imax), ehokardiografskim parametrima veličine infarkta i remodelovanja leve komore tokom hospitalizacije i nakon 6 meseci praćenja (EDVI, ESVI, LVEF, WMSI), i učestalošću neželjenih kardiovaskularnih i cerebralnih događaja (MACCE) tokom perioda praćenja od 6 meseci nakon STEMI (smrt, reinfarkt miokarda, moždani udar, revaskularizacija infarktne arterije, tromboza stenta, hospitalizacija zbog srčane insuficijencije)...Coronary thrombus obtained by manual aspiration thrombectomy from patients with acute ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI) represents convenient material for studying pathophysiologic mechanisms underlying coronary thrombosis, duration of ischemia in STEMI patients, and the degree of thrombus angiogenesis. Vascular endothelial growth factor (VEGF) is the key molecule involved in angiogenesis, which can be measured in the blood of STEMI patients. Level of circulating VEGF could reflect compensatory angiogenic response of ischemic myocardium and represent an important prognostic factor for patients with myocardial infarction. Aim. The aim of this study was to determine the age and the angiogenesis level of aspirated thrombi, and to measure the level of circulating serum VEGF (sVEGF) in STEMI patients treated with pPCI with aspiration thrombectomy, exploring their impact of angiographic and clinical outcomes of STEMI patients. Methods. Study included 100 consecutive STEMI patients with successful thrombectomy during pPCI obtaining adequate amount of material for further pathohistologic analysis (>1 mm3). Aspirated thrombotic material was analyzed by light and confocal microscopy. Age of the aspirated thrombus (fresh, lytic and organized) was determined by hematoxylin-eosin staining. The degree of angiogenesis was assessed by immunohistochemical staning using endothelial cell (EC) markers (CD34 and CD31), and graded as single EC, clusters and EC microvessels. sVEGF was measured during the hospital admission before pPCI and detected by ELISA. Angiographic outcomes were defined as myocardial blush grade (MBG) on final angiogram, with assessing the degree of resolution of ST-elevation (STR) on electrocardiogram 60 minutes after pPCI. Clinical outcomes were evaluated by the final infarct size estimated by the maximum levels of released cardiospecific enzymes during hospitalization (CKmax, troponin Imax), echocardiographic parameters during the initial hospitalization and after 6-months follow-up (EDVI, ESVI, LVEF and WMSI), and the incidence of major adverse cardiovascular and cerebrovascular events (MACCE) during the 6-months follow-up (death, reinfarction, stroke, target vessel revascularization, stent thrombosis, hospitalization due to heart failure)..