Dissertations / Theses on the topic 'Pigment epithelium-derived factor'

Pigment Epithelium-derived Factor (PEDF), a natural protein possessing both neuroprotective and anti-angiogenic properties, is a very unique and attractive candidate as a therapeutic agent in the management of pathological neovascular diseases, such as tumours, age-related macular degeneration (AMD) and diabetic retinopathy. While it is well-known that PEDF can exert powerful effects on various tissues and cells, the underlying mechanism of PEDF's action is not well understood. This study investigated the relationship between vascular endothelial growth factor (VEGF)/PEDF and VEGFR-1 /VEGFR-2 by exploring Presenilin-l (PS-l) dependent regulated intramembrane proteolysis (RIP). Work on this non-classical pathway was initiated by Cai et al., (2006) using in vitro models of bovine retinal microvascular endothelial cells (BRMECs). Current study used BRMECs and human retinal pigment epithelial (HRPE) cells. In this study, BRMECs and HRPE cells were isolated and cultured. BRMECs were used as an angiogenic cell type while HRPE cells were used as an angiogenic regulator cell type. The characteristics of endothelial and epithelial cells and the localisation of VEGFR-1, VEGFR-2 and PS in BRMECs and HRPE cells were determined using immunocytochemistry techniques. The effects of VEGF and PEDF on VEGFR-1, VEGFR-2 and PS were assessed using immunocytochemistry and Western blotting, y-secretase activity in BRMECs and HRPE cells treated with various growth factors were analysed using a y-secretase activity kit. The role of VEGF on the production of PEDF and the expression of VEGFR-1, VEGFR-2 and PS in HRPE cells was investigated at both the transcriptional and translational levels. The techniques, VEGF-small interfering ribonucleic acid (VEGF-siRNA), reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and Enzyme-linked immunosorbent assay (ELISA) were used for the investigation. Results obtained from the project showed that PEDF had a regulatory role in the counterbalance of VEGFR-1 and VEGFR-2 expression in cultured BRMECs. PEDF upregulated y-secretase activity and PS-1 expression in BRMECs while VEGF acted as an antagonist of the effect of PEDF. In contrast, in HRPE cells, VEGF upregulated y-secretase activity and PEDF acted as an antagonist of the effect of VEGF. VEGF-siRNA induced a reduction of PEDF at both transcriptional and protein levels and a reduction of VEGFR-1 at the protein level. The effects of VEGF and PEDF on VEGFR-1 and VEGFR-2 may be cell type dependent. This study strengthens the view that PEDF can exert different regulatory effects on the same molecule (s) in different cell types. PEDF acts either antagonistically to VEGF or synergistically dependent upon the target molecule. Deciphering the cellular and molecular mechanisms underlying these interactions will not only contribute to our understanding of PEDF's action but also provide the foundation to maximise the therapeutic potential of this protein.

There are similarities between tissue repair and cancer development. Epithelial tumours promote the formation of the stroma by activation of the wound healing process, but unlike healing wounds the process is not self-limiting. Pigment epithelium derived factor(PEDF)is a secreted glycoprotein that has been shown to exhibit multiple biological properties including anti-angiogenesis, anti-tumorigenesis and immune-modulation. Previous studies demonstrated that PEDF expression is downregulated as prognostic factors worsen in a range of cancers and chronic inflammatory conditions and treatment with recombinant PEDF has showed some benefit in cellular functional models. However, there has been little evidence to date to assess the role of PEDF in colorectal cancer and wound healing. The aims of this study were to elucidate more detailed regulatory mechanisms of PEDF in wound healing, and tumour angiogenesis in colorectal cancer and provide evidence to develop PEDF or its fragments as therapeutics for wound healing or colorectal cancer treatment. This study found that PEDF expression was down regulated in colorectal cancer cell lines and tissue and that treatment with recombinant PEDF resulted in significant decreases in the rate of colorectal cancer cellular migration and invasion and an increase in cellular adhesion in some colorectal cancer cell lines examined, suggesting a promising role of the treatment of PEDF in the prevention of colorectal cancer metastatic spread. Within wound healing, our results indicated that PEDF expression was high amongst dermal fibroblasts but less so in keratinocytes and endothelial cell lines, suggesting that fibroblasts are responsible for secretion of PEDF in response to inflammation. In cellular functional models, recombinant PEDF treatment significantly increased the migration of keratinocytes, suggesting a possible role as a chronic wound treatment. Further studies are warranted to assess the role of PEDF in a range of colorectal cancer subsets, other wound healing cells and animal models to identify a suitable delivery vector.

BACKGROUND: Prostate cancer is the most common cancer among Swedish men. For patients with metastatic prostate cancer the standard therapy is castration, a treatment that initially provides symptomatic relief but unfortunately is not curative. New therapeutic targets for advanced prostate cancer are therefore needed.  Prostate cancers are composed of tumor epithelial cells as well as many non-epithelial cells such as cancer associated fibroblasts, blood vessels and inflammatory cells.  Many components of the tumor microenvironment such as tumor associated macrophages and angiogenesis have been shown to stimulate tumor progression. This thesis aims to explore mechanisms by which the local environment influences prostate tumor growth and how such mechanisms could be targeted for treatment. MATERIALS AND METHODS: We have used animal models of prostate cancer, in vitro cell culture systems and clinical materials from untreated prostate cancer patients with long follow up. Experiments were evaluated with stereological techniques, immunohistochemistry, western blotting, quantitative real-time PCR, PCR arrays and laser micro dissection. RESULTS: We found that the presence of a tumor induces adaptive changes in the surrounding non-malignant prostate tissue, and that androgen receptor negative prostate tumor cells respond to castration treatment with temporarily reduced growth when surrounded by normal castration-responsive prostate tissue. Further, we show that macrophages are important for prostate tumor growth and angiogenesis in the tumor and in the surrounding non-malignant tissue. In addition, the angiogenesis inhibitor Pigment epithelium-derived factor (PEDF) was found  to be down-regulated in metastatic rat and human prostate tumors. Over-expression of PEDF inhibited experimental prostate tumor growth, angiogenesis and metastatic growth and stimulated macrophage tumor infiltration and lymphangiogenesis. PEDF was found to be down-regulated by the prostate microenvironment and tumor necrosis factor (TNF) α. CONCLUSIONS: Our studies indicate that not only the nearby tumor microenvironment but also the surrounding non-malignant prostate tissue are important for prostate tumor growth. Both the tumor and the surrounding non-malignant prostate were characterized by increased angiogenesis and inflammatory cell infiltration. Targeting the surrounding prostate tissue with castration, targeting tumor associated macrophages, or targeting the vasculature directly using inhibitors like PEDF were all shown to repress prostate tumor growth and could prove beneficial for patients with advanced prostate cancer.

Retinopathy of prematurity, a disease characterised by aberrant retinal vascular development in premature neonates, is a leading cause of blindness and visual impairment in childhood. This work sought to examine differences in the susceptibility of inbred rat strains to oxygen-induced retinopathy, a model of human retinopathy of prematurity. The overriding aim was to identify genetic factors in rats that might be generalisable to humans. Newborn rats of six different strains were exposed to alternating cycles of hyperoxia and relative hypoxia for fourteen days. Rats were removed to room air and killed for analysis immediately, to assess oxygen-induced retinal vascular attenuation, or four days later to evaluate the extent of hypoxia-induced vasoproliferation. Whole flat-mounted retinae were stained with fluorophore conjugated isolectin GS-IB4, and measurement of vascular area was conducted using fluorescence microscopy and video-image analysis. A hierarchy of susceptibility to the inhibitory effects of cyclic hyperoxia and relative hypoxia on postnatal retinal vascularization was identified for the rat strains studied. Susceptibility to vascular attenuation was predictive of the subsequent risk of vascular morphological abnormalities. Cross-breeding experiments between susceptible and resistant strains demonstrated that the susceptible phenotype was dominantly inherited in an autosomal fashion. These studies confirmed an association between ocular pigmentation and retinopathy risk, however the finding of differential susceptibility amongst albino rat strains implicated factors in addition to those associated with ocular pigmentation. Quantitative real-time reverse transcription-polymerase chain reaction was used to compare the retinal expression of angiogenic factor genes in susceptible and resistant strains with the aim of identifying a genetic basis for the strain difference. Eight angiogenic factor genes were selected for study: vascular endothelial growth factor (VEGF); VEGF receptor 2; angiopoietin 2; Tie2; pigment epithelium-derived factor; erythropoietin; cyclooxygenase-2 and insulin-like growth factor-1. The most notable difference between strains was the expression of vascular endothelial growth factor (VEGF) during the cyclic hyperoxia exposure period - higher VEGF expression was associated with relative resistance to retinopathy. Other differences in retinal angiogenic factor gene expression between strains, such as higher expression of VEGF receptor 2 and angiopoietin 2 in resistant strains, appeared to be secondary to those in VEGF. Following cyclic hyperoxia, the expression pattern of angiogenic factor genes changed - messenger RNA levels of hypoxia-induced genes, including VEGF, VEGF receptor 2, angiopoietin 2 and erythropoietin, were significantly higher in those strains with larger avascular areas, than in those strains that were relatively resistant to retinopathy. These findings provide firm evidence for hereditary risk factors for oxygen-induced retinopathy in the rat. Differences in the regulatory effects of oxygen on VEGF expression appear to be central to the risk of retinopathy. The potential relevance of these hereditary factors is discussed in the context of the human disease.

La degeneración macular asociada a la edad (DMAE) es una de las principales causas de ceguera legal en adultos de países desarrollados. Actualmente, todos los tratamientos disponibles para la DMAE exudativa están dirigidos a detener la pérdida de la visión, no habiendo tratamientos que puedan revertir el daño. La progresión de la DMAE exudativa depende de la disregulación entre factores pro- y anti- angiogénicos, con un aumento de los niveles de VEGF y una disminución de los niveles de PEDF. Desarrollar una terapia no invasiva y eficaz para el tratamiento de la DMAE húmeda basada en una fracción activa de PEDF (faPEDF), permitiría mejorar la seguridad y comodidad de aplicación del tratamiento abriendo una nueva vía terapéutica. El objetivo principal de este proyecto es evaluar la eficacia de la administración de una faPEDF sintética por vía tópica con el fin de determinar una nueva vía terapéutica para la neovascularización coroidea (NVC) secundaria a DMAE. En una primera fase in vitro se determinará el efecto antiangiogénico de las diferentes faPEDF mediante ensayos de migración celular, formación tubular y proliferación celular en cultivos primarios de células endoteliales. En una segunda fase in vivo se desarrollará un modelo animal de NVC en ratones como modelo plausible de NVC asociada a DMAE, se valorará el efecto de la administración tópica de la faPEDF en el control de las lesiones neovasculares en el modelo animal y se evaluaran los efectos antiinflamatorios, antiangiogénicos y neurotróficos en la retina de ratón tratada con la faPEDF. Las faPEDF2 y faPEDF3 se mostraron eficaces inhibiendo los efectos del VEGF, la formación tubular, la migración y proliferación celular. La realización de láser verde 532nm con parámetros de 250mW de potencia, 100ms de duración y 50 micras de tamaño de spot fue el modelo más adecuado y reproducible de NVC en ratón. Finalmente, la administración diaria de la faPEDF3 en forma de colirio a una concentración de 1 mg/ml en ratones causó una mayor reducción de la superfície de NVC respecto al placebo y también provocó una disminución en la expresión de VEGF en los ojos tratados respecto al placebo.
Age related macular degeneration (AMD) is a leading cause of legal blindness in adults in developed countries. Currently, all available treatments for wet AMD are aimed at stopping the loss of vision, having no treatment that can reverse the damage. The progression of exudative AMD depends on the dysregulation between pro and anti-angiogenic factors, with increased levels of VEGF and decreased levels of PEDF. Developing a noninvasive and effective therapy for the treatment of wet AMD based on an active fraction of PEDF (afPEDF) would improve the safety and comfort of applying the treatment, enabling a new therapeutic approach. The main goal of this project is to evaluate the effectiveness of the administration of a synthetic afPEDF topically in order to determine a new therapeutic approach for choroidal neovascularization (CNV) secondary to AMD. In a first stage, in vitro assays will be performed to determ the antiangiogenic effect of the different afPEDF by cell migration assays, cell proliferation and tube formation in primary cultured endothelial cells. In a second phase, CNV will be developed in vivo in an animal model of mice as a plausible model of CNV associated with AMD. The effect of topical administration of afPEDF among neovascular lesions in the animal model will be assessed, and inflammatory, angiogenic and neurotrophic effects in the retina of mice treated with faPEDF will be also evaluated. Both afPEDF2 and afPEDF3 were effective in inhibiting the effects of VEGF, tube formation, migration and cell proliferation. Performing 532nm green laser with parameters of 250 mW, 100 ms duration and 50-micron spot size was the most suitable and reproducible model of CNV in mice. Finally, daily administration of afPEDF3 drops at a concentration of 1 mg / ml in mice caused a greater reduction in the area of CNV over placebo and also caused a decreased expression of VEGF treated eyes when compared to placebo.

碩士
國立臺灣大學
微生物學研究所
95
The hepatocellular carcinoma is considered as one of the suitable targets for anti-angiogenic approaches due to its highly neovascularization. However, anti-angiogenic therapies aimed at single target can be neutralized by up-regulation of other pro-angiogenic factor. The fact that redundant angiogenic factor can be up-regulated by tumors in advanced stage of carcinoma suggests that the angiostatic strategies may require a combination of multiple anti-angiogenic factor. In this study, we assessed the therapeutic effects of combination therapy with two anti-angiogenic factors, Endostatin (ED) and Pigment Epithelium-Derived Factor (PEDF), on established orthotopic liver tumor. We used recombinant adenoviral vector carrying genes encoded mouse ED or human PEDF. The adenovirally expressed endostatin and PEDF were biologically active, as demonstrated in vitro and in vivo. We performed an in situ tumor therapy with co-administration of ED and PEDF by recombinant adenoviral vectors. The results of animal experiments indicated that the combination of both factors provided a synergistic effect on tumor regression. The immunohistochemistry staining of endothelial cell marker in tumor regions also revealed that the combined therapy synergistically reduced the tumor vascularization. The inhibition of tumor vascularization which starved tumors from oxygen and nutrients induced apoptosis in tumor regions. Furthermore, we examined the impact of both factors on Vascular Endothelial Growth Factor (VEGF) mRNA level. A down-regulation of VEGF mRNA level was observed in response to ED or PEDF treatment. Moreover, the combined ED/PEDF treatment was able to augment the inhibition of VEGF expression. Surprisingly, the combination therapy also induced a higher level of tumor-infiltrating CD8+ T lymphocytes. We infer that the apoptosis induced by anti-angiogenesis gave rise to a bystander effect which activated higher levels of CD8+ T cells. Based on this hypothesis, we combined immunotherapy and anti-angiogenic therapy to improve the therapeutic effect on orthotopic hepatocellular carcinoma. We performed a combination of immunotherapy and anti-angiogenic therapy by adenoviruses-mediated gene transfer of ED, PEDF, IL-12, and GM-CSF simultaneously on the orthotopic hepatocellular carcinoma. The “four-in-one” therapy exhibited a synergistic effect on tumor regression compared to the immunotherapy group (GM-CSF + IL-12) or anti-angiogenic group (ED + PEDF) at the same dose. Our findings suggest that the co-action between two different treatment strategies can more effectively reduce tumor burden compared to single treatment strategy. Hence, the combination strategy between immunotherapy and anti-angiogenic therapy renders a promising treatment for hepatocellular carcinoma.

碩士
國立臺灣大學
解剖學暨細胞生物學研究所
104
Obesity is the modern disaster of human being, and is the major cause of many metabolic diseases and cardiovascular diseases. It presents with metabolic disorders such as hyperglycemia, hyperlipidemia, and insulin resistance. Previous studies reveal that the oxidative stress and inflammation in the adipose tissues take the responsibility of metabolic diseases, and they also concentrate in the differentiation of adipocytes and the dynamic balance of lipolysis. Pigment epithelium-derived factor (PEDF), a well-known cytokine which isolate from retina pigment epithelial cells, has the multiple function of potential neuronal differentiating activity, anti-neoplasm, smooth muscle growth inhibition, and anti-inflammation. Further studies also demonstrate that the adipose tissue is not only the source of PEDF, but also the target of this unique protein. The aim of this study is to investigate the role of PEDF to inhibit obesity and anti-adipogenesis. In vivo, we feed C57BL/6 mice with high fat (HF) diet as animal model. The animals are divided into HF fed with PEDF group and DIO (diet-induced obesity) with PEDF group, compared with control group, to study the anti-obesity effect of PEDF. In vitro, we use 3T3-L1 preadipocyte as cell model, and treat with PEDF during differentiation and after differentiation. By lipid droplet formation, we inspect the inhibition effect of PEDF to adipocyte differentiation and lipid metabolism such as lipolysis and lipogenesis. In animal study, the data shows that PEDF effectively decreases body weight gain, suppresses and slow down the obesity, decreases white adipose tissue (WAT) formation and inflammation. It also improves insulin resistance, hyperglycemia and decrease serum lipid. In brown adipose tissue (BAT), PEDF decreases the lipid accumulation. In liver, our study proved that PEDF decreases lipid formation and fibrosis. In vitro study, PEDF diminishes lipid droplet formation after 3T3-L1 differentiations. We also found out that PEDF prolongs the cell cycle progress and promotes lipolysis, which may work through mTOR-S6K pathway and subsequent transcription factors such as PPAR-γ, CEBP-α, and CEBP-β. These findings prove the hypothesis that PEDF diminishes white adipocyte formation, promotes lipid metabolism, and subsequent weight loss in mice. In conclusion, PEDF protects against high-fat diet-induced obesity and metabolic disorders in mice, and inhibits adipocyte differentiation in 3T3-L1 cells. It provides a new treatment trend for obesity in the future.

Yes
Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods P EDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell cocultures and matrigel assays were employed to assess angiogenesis. Results P EDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions O ur data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.
NIHR CDF; EULAR ODP

博士
國立臺灣大學
解剖學暨生物細胞學研究所
97
The number of endothelial progenitor cells (EPCs) that can be obtained from adult bone marrow and peripheral blood to treat cardiovascular diseases is limited. The goal was to examine the endothelial potential of Wharton’s jelly in human umbilical cord (WJC)-derived stem cells and evaluate their potential to affect neointimal formation after vascular injury. Mesenchymal cells (MCs) were isolated from WJC and cultured in endothelial growth medium. Differentiation into late outgrowth endothelial cells (WJC-OECs) was demonstrated by incorporation of acetylated low-density lipoprotein and expression of the endothelial-specific markers. Transplantation of these cells into wire-injured femoral arteries in mice led to rapid re-endothelialization. At 4 weeks after injury, the neointima/media area ratio was reduced and strong expression of pigment epithelium-derived factor (PEDF) compared to saline- or MC- or cord blood-OEC- treated mice. WJC-OECs-conditioned medium has an extremely strong capacity to inhibit the migration and proliferation of smooth muscle cells. The effects were inhibited by neutralizing antibody for PEDF and by siRNA silencing of PEDF. We firstly demonstrated the presence of a cell population within WJC that has the potential to differentiate into OECs. Transplantation of WJC-OECs may play a crucial role in reestablishing endothelial integrity in injured vessels, thereby inhibiting neointimal hyperplasia. These findings have implications for a novel and practical cell-based therapy for vascular diseases.

Stroke, after myocardial infarction and cancer is the third most common cause of death worldwide and 1/6th of all human beings will suffer at least one stroke in their lives. Furthermore, it is the leading cause for adult disability with approximately one third of patients who survive for the next 6 months are dependent on others. Because of its huge socioeconomic burden absorbing 6% of all health care budgets and with the fact that life expectancy increases globally, one can assume that stroke is already, and will continue to be, the most challenging disease. Ischemic stroke accounts for approximately 80% of all strokes and results from a thrombotic or embolic occlusion of a major cerebral artery (most often the middle cerebral artery, MCA) or its branches Following acute ischemic stroke, the most worrisome outcome is the rapidly increasing intra-cranial pressure due to the formation of space-occupying vasogenic oedema which can have lethal consequences. Permeability changes at the Blood-Brain Barrier (BBB) usually accompanies the oedematous development and their time course can provide invaluable insight into the nature of the insult, activation of compensatory mechanisms followed by long term repair. Rodent models of focal cerebral ischemia have been developed and optimized to mimic human stroke conditions and serve as indispensable tools in the field of stroke research. The presented work constituting of three separate but complete works by themselves are sequential, where, the first part was dedicated to the establishment of non-invasive small animal imaging strategies on a 3 tesla clinical magnetic resonance scanner. This facilitated the longitudinal monitoring of pathological outcomes following stroke where identical animals can serve as its own control. Tissue relaxometric estimations were carried out initially to derive the transverse (T2), longitudinal (T1) and the transverse relaxation time due to magnetic susceptibility effects (T2*) at the cortical and striatal regions of the rodent brain. Statistically significant differences in T2*-values could be found between the cortex and striatal regions of the rodent brain. The derived tissue relaxation values were considered to modify the existing imaging protocols to facilitate the study of the rodent model of ischemic stroke. The modified sequence protocols adequately characterized all the clinically relevant sequels following acute ischemic stroke, like, the altered perfusion and diffusion characteristics. Subsequent to this, serial magnetic resonance imaging was performed to investigate the temporal and spatial relationship between the biphasic nature of BBB opening and, in parallel, the oedema formation after I/R injury in rats. T2-relaxometry for oedema assessment was performed at 1 h after ischemia, immediately following reperfusion, and at 4, 24 and 48 hours post reperfusion. Post-contrast T1-weighted imaging was performed at the last three time points to assess BBB integrity. The biphasic course of BBB opening with significant reduction in BBB permeability at 24 hours after reperfusion was associated with a progressive expansion of leaky BBB volume, accompanied by a peak ipsilateral oedema formation. At 48 hours, the reduction in T2-value indicated oedema resorption accompanied by a second phase of BBB opening. In addition, at 4 hours after reperfusion, oedema formation could also be detected at the contralateral striatum which persisted to varying degrees throughout the study, indicative of widespread effects of I/R injury. The observations of this study may indicate a dynamic temporal shift in the mechanisms responsible for biphasic BBB permeability changes, with non-linear relations to oedema formation. Two growth factor peptides namely pigment epithelium derived factor (PEDF) and epidermal growth factor (EGF) with widely different trophic properties were considered for their beneficial effects, if any, in the established rodent model of I/R injury and studied up to one week employing magnetic resonance imaging. Both the selected, trophic factors demonstrated significant neuroprotection as demonstrated by a reduction in infarct volume, even though PEDF was found to be the most potent one. PEDF also demonstrated significant attenuation of oedema formation in comparison to both the control and EGF groups, even though EGF could also demonstrate oedema suppression. In the present work, we noticed that interventions with macromolecule protein/peptides by itself could mediate remote oedema at distant sites even though the significance of such an observation is not clear at present. Susceptibility (T2*) weighted tissue relaxometric estimations were considered at the infarct region to detect any metabolic changes arising out of any neuroprotection and/or cellular proliferation / neurogenesis. PEDF group demonstrated a striking reduction of the T2*-values, which is indicative of an increased metabolic activity. Moreover, all the groups (Control, EGF and PEDF) demonstrated significantly elevated T2*-values at the contralateral striatum, which is indicative of widespread metabolic suppression usually associated with a variety of traumatic brain conditions. Moreover, as expected from the properties of PEDF, it demonstrated an extended BBB permeability suppression throughout the duration of the study. This study underlines the merits of considering non-invasive imaging strategies without which it was not possible to study the required parameters in a longitudinal fashion. All the observations are adequately supported by reasonably well defined mechanisms and needs to be further verified and confirmed by an immunohistochemical study. These results also need to be complemented by a functional study to evaluate the behavioural outcome of animals following these treatments. These studies are progressing at our laboratory and the results will be duly published afterwards
Schlaganfall ist nach Herzinfarkt und Krebs die dritthäufigste Todesursache weltweit und 1/6 aller Menschen erleiden mindestens einen Schlaganfall in ihrem Leben. Wichtiger ist jedoch, dass Schlaganfallerkrankungen ist die führende Ursache für dauerhafte Behinderungen darstellen. Ungefähr ein Drittel dieser Patienten, die die ersten 6 Monate überleben sind auf die Hilfe anderer angewiesen. Die enorme Wichtigkeit des Schlaganfalls begründet sich zudem dadurch, dass schon jetzt die sozioökonomischen Kosten 6% der gesamten Gesundheitausgaben betragen und die globale Lebenserwartung weiter steigen wird. Der ischämische Hirninfarkt ist der mit 80% vorherrschende Schlaganfalltyp und resultiert aus thrombotischen und embolischen Verschlüssen der großen hirnversorgenden Arterien und deren Ästen (insbesondere der A. cerbri media). Die wichtigste Komplikation mit hoher Mortalität nach einem ischämischen Schlaganfall ist Entwicklung eines raumfordernden vasogene Ödementwicklung. Änderungen der Permeabilität der Blut-Hirn Schranke (BHS) begleitet die Ödementwicklung und Analyse der zeitlichen BHS Permeabilität können wichtige Erkenntnisse über den natürlichen Verlauf eines Hirninfarkts und die Aktivierung kompensatorischer Mechanismen, die in Reparaturvorgänge münden, liefern. Verschiedene modelle des akuten ischämischen Schlaganfalls mit Nagetieren wurden entwickelt um die Schlaganfalltherapie des Menschen weiter zu entwickeln. Sie sind momentan unersetzliche Instrumente in der Schlaganfallforschung. Die hier vorgestellte Arbeitet setzt sich auch 3 abgeschlossenen sequentiellen Teilprojekten zusammen. Das erste Teilprojekt befasst mit der Etablierung von der nicht-invasiven Kleintierbildgebung auf einem klinischen 3 Tesla Kernspintomographen. Diese Arbeit bildet die Grundlage für das in vivo Monitoring der pathologischen Veränderungen nach Schlaganfall in einem identischen Versuchstier nachverfolgt werden kann und so die eigene Kontrolle darstellt. Gewebs relaxometrische Messungen wurden initial durchgeführt um die transverse (T2), longitudinal (T1) und transverse (T2*) Relaxationszeit (aufgrund der magnetischen Suszeptibilitätseffekte) in kortikalen und striatalen Hirnregionen der Nagetiere zu bestimmen. Statistisch signifikante Unterschiede in der T2*- Werte konnte zwischen der kortikalen und Striatalen Regionen des Gehirn von Nagetieren gefunden werden. Hierdurch konnten bestehende Messprotokolle vom Menschen auf die Nagetiere optimiert und für die Untersuchungen des Schlaganfalls genutzt werden. Diese Vorarbeiten erlauben klinisch relevante Veränderungen wie eine veränderte Diffusion und Perfusion nach ischämischen Schlaganfall zu verfolgen. Basierend auf diesen Vorarbeiten wurde die örtliche und zeitliche Charakterisierung der bi-phasischen BHS-Öffnung und der Ödementwicklung nach experimenteller I/R der Ratte mittels serieller Magnetresonanztomographie (MRT) untersucht. Hier diente die T2-Relaxometrie zur Ödemquantifizierung und wurde 1 Stunde nach Beginn der zerebralen Ischämie, unmittelbar nach Reperfusion und im Intervall von weiteren 4, 24 und 48 Stunden durchgeführt. Eine T1-gewichtete Sequenz wurde vor und nach Gabe von Kontrastmittel an den drei letztgenannten Zeitpunkten zeigte den bi-phasischen Verlauf der BHS-Öffnung 4 und 48 Stunden nach Reperfusion. Eine signifikante Reduktion der BHS Permeabilität 24 Stunden war zum einem mit einer Erhöhung des Gesamtvolumens der gestörten BHS und zum anderen mit Maximum der Ödementwicklung assoziiert. Darüber hinaus konnte 48 Stunden nach Reperfusion bereits eine Resorption des Ödems anhand der T2-Relaxometrie gemessen werden während die zweite Phase der bi-phasischen BHS-Öffnung auftrat. Zusätzlich trat 4 Stunden nach Reperfusion eine Ödembildung auch der nicht-ischämischen Striatum auf, welche in unterschiedlichem Maße über die Studiendauer persistierte. Dies spricht dafür, dass Ischämie und Reperfusion Effekte auf das gesamte Gehirn haben können. Zusammenfassend sprechen die Beobachtung dafür, dass der zeitlichen Entwicklung des Hirnödems verschiedene Mechanismen der erhöhten Blut-Hirn Schranken Permeabilität zu Grunde liegen. Zwei Wachstumsfaktoren, der, Pigment epithelium abstammende Wachstumsfaktor (PEDF) und der epidermale Wachstumsfaktor (EGF), mit deutlich unterschiedlichen trophischen Eigenschaften wurden auf ihre positiven Effekte im etablierten Tiermodel der zerebralen I/R hin untersucht. Dabei wurden serielle MRT Untersuchungen bis hin zu einer Woche genutzt. Beide Wachstumsfaktoren führten im Model zu einer signifikanten Neuroprotektion, die sich in einer Reduktion des Infarktvolumens gegenüber einer Kontrolle mit Kochsalz zeigte. PEDF allerdings hatte gegenüber EGF eine potentere Wirkung und zeigte darüber hinaus und noch deutlicher als EGF eine signifikante Verminderung der Ödembildung. Allerdings zeigte eine Behandlung mit diesen großmolekularen Proteinen zumindest nach 24 Stunden auch eine Neigung zur Ödembildung in vom Schlaganfall nicht betroffenen Hirnarealen, deren Signifikanz allerdings noch unklar ist. T2*- gewichtete Relaxationsmessungen können dazu genutzt werden, um metabolische Veränderungen, die aus neuroprotektiven Therapieansätzen bzw. zellulärer Proliferation bzw. Neurogenese entstehen, zu quantifizieren. Hier zeigten insbesondere mit PEDF behandelte Versuchstiere eine hochsignifikante Reduktion der T2*-Werte, welche als Hinweis auf eine erhöhte metabolische Aktivität gewertet werden können. Insgesamt zeigten alle Behandlungsgruppen (Kochsalzkontrollen, EGF und PEDF) behandelte Tiere signifikant erhöhte T2*-werte auf des kontralateralen Striatums, welche auf eine weitreichende metabolische Suppression hindeuten, wie sie normalerweise bei einer Reihe von traumatischen Hirnerkrankungen gefunden werden können. Ein weiterer Befund ist, wie erwartet, die ausgedehnte Verminderung der BHS-Durchgängigkeit durch PEDF über die gesamte Dauer der Untersuchung hinweg. Diese Studie unterstreicht den Nutzen nicht-invasiver Bildgebungsstrategien, ohne die die Untersuchung der benötigten Parameter in einem longitudinalen Design nicht möglich wäre. Ausblickend müssen diese gut mittels MRT charakterisierten Prozesse durch immunhistologische und funktionelle Untersuchungen gestützt und nachfolgend publiziert werden