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The peroxisome proliferator activated receptor gamma (PPARgamma) plays an eminent role during alternative activation of macrophages and resolution of inflammation. As an antiinflammatory signaling molecule, it seems likely that it is tightly regulated dependent on the state of the immune response. There is growing evidence that PPARgamma expression is reduced during inflammation, whereas molecular mechanisms are illdefined. Even though, its role in immunosuppression is getting more definite. Apoptotic cells (AC) provoke an active repression of pro-inflammatory responses inter alia by the inhibition of pro-inflammatory cytokine expression or attenuated generation of reactive oxygen species (ROS). The reduced formation of ROS was attributed to PPARgamma activation, while mechanisms behind the reduced cytokine expression remained unclear. Therefore, my Ph.D. thesis addressed the role of PPARgamma during inhibited cytokine synthesis in response to AC and the regulation of PPARgamma expression during an inflammatory response, which was initiated by lipopolysaccharide (LPS) exposure. In the first part of the thesis, I investigated the role of PPARgamma in coordinating the attenuation of pro-inflammatory cytokine expression in response to AC. Exposing murine RAW264.7 macrophages to AC prior to LPS-stimulation, reduced NFKB transactivation and lowered target gene expression of e.g. TNFalpha and IL-6 compared to controls. In macrophages over-expressing a dominant negative (d/n) mutant of PPARgamma, NFKB transactivation in response to LPS was restored, while using macrophages from myeloid lineage-specific conditional PPARgamma knock-out mice proved that PPARgamma transmitted the anti-inflammatory response delivered by AC. Domain analysis revealed that amino acids 32-250 are essential for inhibition of NFKB. Mutation of a SUMOylation (SUMO: small-ubiquitin related modifier) site in this region (K77R) and interfering SUMOylation by silencing the SUMO E3 ligase PIAS1 (protein inhibitor of activated Stat1) eliminated AC-provoked NFKB inhibition and concomitant TNFalpha expression. Chromatin-immunoprecipitation assays demonstrated that AC prevented the LPS-induced removal of nuclear receptor co-repressor (NCoR) from the KB response element within the TNFalpha promoter. I concluded that AC induce PPARgamma SUMOylation to attenuate the removal of NCoR, thereby blocking transactivation of NFKB. This contributes to an anti-inflammatory phenotype shift in macrophages in response to AC, by lowering pro-inflammatory cytokine production. The second part addressed molecular mechanisms responsible for reduced PPARgamma expression upon LPS exposure. PPARgamma gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer’s disease is associated with impaired PPARgamma expression. Initiation of an inflammatory response by exposing primary human macrophages to LPS revealed a rapid decline of PPARgamma1 expression. PPARgamma1 mRNA decrease was prevented by inhibition of NFKB and also after pre-treatment with the PPARgamma agonist rosiglitazone, suggesting a NFKB-dependent pathway, because activated PPARgamma is known to inhibit NFKB transactivation. Since promoter activities were not affected by LPS, I focused on mRNA stability and noticed a decreased PPARgamma1 mRNA half-life. RNA stability is often regulated via 3’ untranslated regions (UTRs). Therefore, I analyzed the impact of the PPARgamma-3’UTR by luciferase assays. LPS significantly reduced luciferase activity of pGL3-PPARgamma-3’UTR, suggesting that PPARgamma1 mRNA is destabilized. Deletion of a potential miR-27a/b binding site within the 3’UTR completely restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS-exposure, partially reversed PPARgamma1 mRNA decay, whereas the mature miR-27 mimicked the effect of LPS. MiR-27b was at least partially induced by NFKB, thus correlating with NFKB-dependent PPARgamma1 mRNA decrease. Since deletion of the miR-27 site also containing an AU-rich element (ARE) completely abrogated LPS-induced reduction but inhibition of miR-27b only partially restored PPARgamma1 mRNA expression, I suggested an additional implication of an ARE-binding protein. I provide evidence that LPS induces miR-27b, which in turn destabilizes PPARgamma1 mRNA. Understanding the molecular mechanism of PPARgamma mRNA destabilization, might help to rationalize inflammatory diseases associated with impaired PPARgamma expression. Even though, further experiments are needed to clarify the potential involvement of ARE-binding proteins.
It has been shown that stem and progenitor cells are therapeutically effective after i.v application. Yet, many aspects regarding intracellular signaling pathways which are involved in the homing and local action of these cells still have to be elucidated. In this work, it was aimed to investigate the role of the small GTPase Rap1 in adhesion activation in Hematopoietic Stem and Progenitor Cells (HSC/HPC) and in Mesenchymal Stem Cells (MSC). The potential role of Rap1 was assessed in, mice which were homozygote negative for the expression of the Rap1a gene. Peripheral blood lymphocyte counts as well as numbers of HPCs in the blood were decreased in Rap1a-/- mice compared to wild-type controls. Additionally the adhesion capability of HPCs from Rap1a-/- to the endothelial ligand, Vascular Cell Adhesion Molecule – 1 under shear stress was decreased. The hematopoietic repopulation potential of Rap1a-/- HPC was however not decreased in a competitive bone marrow transplantation model, indicating that deficiency of Rap1a in HSC/HPC does not negatively affect their ability to interact with the bone marrow microenvironment. In contrast, the isolation of MSC was not possible from Rap1a-/- bone marrow, indicating an altered situation in the bone marrow niche through changed stromal cell behaviour. Instead, Rap1a+/- MSC could be isolated and showed an adhesion deficit under shear stress. In contrast, no differences were noted in their differentiation potential. In a mouse homing model, the overall ability of the Rap1a+/- MSC to home to different tissues was found preserved. Finally, in a murine subcutaneous carcinoma model, cells with an HPC phenotype were observed to be present in the tumor microenvironment, and it was shown that they home directly to tumors. Since HPC isolated from bone marrow were able to differentiate into cells with a pro-angiogenic phenotype in vitro, HPC may be of relevance for neovascularization, as tumor-infiltrating progenitor cells. The results of the study should contribute to the understanding of the regulation of progenitor cell homing behaviour in situations simulating cell therapy approaches in preclinical situations.
Sepsis is caused by infection and often followed by an overwhelming inflammatory response. This can lead to shock, organ failure and even death. Each year approximately 60,000 people die in Germany due to sepsis. There is good evidence that sepsis is associated with failure of the hypothalamic-pituitary-adrenal-axis. In patients with sepsis, glucocorticoids (e.g. corticosterone, cortisol) released from adrenal glands play an essential role in preventing an excessive pro-inflammatory response. Adrenal insufficiency occurs in a large number of patients with septic shock and is associated with an increased mortality. In the innate immune system, Toll-like receptors (TLRs) play a crucial role in its onset by recognizing pathogenassociated molecules. It is well known that there are interactions between the immune and endocrine stress systems; glucocorticoids and TLRs regulate each other in a bi-directional way. Therefore, a coordinated response of the adrenal and immune system is of vital importance for survival during severe inflammation. This experimental study focuses on the role of TLR-2, TLR-4 and TLR-9 during adrenal stress. The results show that in mice, the absence of TLR-2 and TLR-4, but not TLR-9 leads to altered adrenal morphology, relating to size and cellular structure. However, this alteration does not appear to compromise the phenotype of TLR knock-out mice. Mice deficient of TLR-2, 4 and 9 are not able to respond adequately to inflammatory stress induced by their potential ligands lipopolysaccharide (LPS), lipoteichoic acid (LTA) or cytidine phosphate guanosine-oligodeoxynucleotides (CpG-ODN). This impaired adrenal stress response appears to be associated with a decrease in systemic and intra-adrenal cytokine expressions. Taken together, these results suggest that TLR-2, 4 and 9 are key players in the immuno-endocrine response during inflammation and SIRS. In conclusion, TLRs play a crucial role in the immune-adrenal crosstalk. This close functional relationship needs to be considered in the treatment of inflammatory diseases where an intact adrenal stress response is required. Furthermore, TLR polymorphisms could contribute to the underlying mechanisms of impaired adrenal stress response in patients with bacterial sepsis
Macrophages show a remarkable functional plasticity, which enables them to change their phenotype in response to environmental signals. They are key players during infection by initiating inflammation through the release of proinflammatory mediators. Furthermore, macrophages contribute to the resolution of inflammation by phagocytosis of apoptotic granulocytes. Phagocytosis of apoptotic cells (AC) induces an anti-inflammatory phenotype in macrophages and protects them against apoptosis. However, mechanistic details provoking these phenotype alterations are incompletely understood. Therefore, the aim of my Ph.D. thesis was to investigate the molecular basis of anti-inflammatory macrophage polarization. In the first part of my studies, I investigated the expression of heme oxygenase (HO)-1 in macrophages following treatment with supernatants from AC. HO-1 catalyzes the first and rate-limiting step of heme degradation and potentially bears anti-inflammatory as well as anti-apoptotic potential. I was able to show biphasic upregulation of HO-1 by AC supernatants. The first phase of HO-1 induction at 6 h required activation of p38 MAPK and was accomplished by the bioactive lipid sphingosine-1-phosphate (S1P) engaging S1P receptor 1 (S1P1). However, the second wave of HO-1 induction at 24 h was attributed to autocrine signaling of vascular endothelial growth factor (VEGF) A, whose expression was facilitated by S1P. The release of VEGFA from macrophages was STAT1-dependent, whereas VEGFA itself acted on the macrophage HO-1 promoter via STAT1/STAT3 heterodimer binding. Knockdown of HO-1 revealed its relevance in promoting enhanced expression of the anti-apoptotic proteins B cell leukemia/lymphoma-2 (Bcl-2) and B cell leukaemia/lymphoma-x long (Bcl-XL), as well as the anti-inflammatory adenosine receptor A2A. MHC II and indoleamine 2,3-dioxygenase expression were also affected by ACsupernanatants, but were not HO-1 dependent. Unexpectedly, S1P1 was also upregulated following treatment with AC supernatants. Thus, I considered whether S1P1 induction could specifically be mediated by alternative macrophage activating factors. The expression of S1P1 was enhanced in the presence of the alternative activation stimuli IL-4 as well as IL-10, whereas it was unchanged following incubations with LPS, interferon-g or S1P. My next aim was to investigate the expression of the different S1P receptor isoforms in macrophages following treatment with supernatants form AC. While the expressions of S1P1 as well as S1P3 were induced by exposure to supernatants from AC, S1P2 expression was unaffected. As S1P1/3 and S1P2 are conflictively involved in the regulation of cell migration, I asked for a correlation between increased S1P receptor expression and enhanced migration rate. Indeed, macrophages showed enhanced motility following treatment with supernatants form AC, which was inhibited in S1P1 knockout macrophages. In summary, my findings indicate that HO-1, which is induced by AC-derived S1P, is critically involved in macrophage polarization towards an alternatively activated macrophage phenotype. S1P1 seems to represent a central checkpoint during macrophage activation. On the one hand, S1P1 is induced by supernatants form AC and promotes migration of macrophages. On the other hand, it mediates the induction of HO-1, which is accompanied by antiinflammatory as well as anti-apoptotic signaling. Furthermore, my studies provide evidence that upregulation of HO-1 and S1P1 in macrophages may contribute to the resolution of inflammation by establishing an anti-inflammatory macrophage phenotype and provoking macrophage migration along the vascular S1P gradient out of an inflammatory environment into the lymph.
In 1911 Eugen Bleuler (Bleuler, 1911) postulated that schizophrenia was a disorder resulting from inability to properly integrate mental processes. Around the same time, Carl Wernicke (Wernicke, 1894) proposed that psychosis might result from disruption of white matter tracts. Both of these statements can be considered early cornerstones of modern connectivity hypotheses developed towards the end of the twentieth century by such researchers as Karl Friston (1998) and Nancy Andreansen (1998). In the current work, the hypothesis that schizophrenia, rather than being a disorder or either anatomical or functional connectivity, is a disorder where both of these processes interact and influence the clinical presentation of patients, is examined. This is achieved through a detailed examination of a sample of chronic schizophrenia patients using a combination of functional and anatomical Magnetic Resonance Imaging techniques. The relationship of these measures to clinical symptoms is also explored. In the first study, anatomical connectivity at the whole-brain level is examined using Diffusion Tensor Imaging. The results of the study contribute to the previous literature on auditory hallucinations in schizophrenia and provide the first direct correlation between increased anatomical connectivity and increased severity of psychotic symptoms. The second study provides a thorough examination of the interhemispheric connectivity. This is achieved through a detailed examination of the corpus callosum using a combination of diffusivity and volumetric values. This is the first study to date where several anatomical methods are used in one sample. The results illustrate the importance of using different techniques to accurately characterize anatomical abnormalities observed in schizophrenia. In addition, contrary to previous research reports, the results of the current study imply that only specific sub-sections of the corpus callosum are affected by anatomical abnormalities. The pattern of these changes may influence clinical presentation of patients. Finally, functional connectivity at the whole-brain level is examined during resting-state using Independent Component Analysis. Similarly to the results of the anatomical examinations, it provides further supporting evidence that the pattern of disturbances observed in the current sample of schizophrenia patients examined herein reflects a combination of hypo- and hyperconnectivity. Moreover, the study further validates resting-state functional Magnetic Resonance Imaging as a reliable tool for examining functional abnormalities in schizophrenia.
Background and objectives: Constrictive pericarditis (CP) is the result of a spectrum of primary cardiac and non-cardiac conditions. Little data exists on the cause-specific survival after pericardiectomy in the modern era. The impact of pericardial calcification (CA) on survival is unclear. We sought to determine the association of etiology of CP, CA and other clinical variables with long-term survival after pericardiectomy. Methods: We analyzed the records of 163 patients who underwent pericardiectomy for CP over a 24-year period at a single center. The diagnosis of CP was established by surgical report. Vital status was obtained by the Social Security Death Index. The Kaplan Meier method was used to estimate overall survival and survival by etiology group. Cox proportional hazards regression analysis was performed to assess the effect of various causes for CP on longterm survival while adjusting for age. Results: The etiology of CP was idiopathic in 75 patients (46%), prior cardiac surgery in 60 (37%), radiation treatment in 15 (9%) and miscellaneous in 13 patients (8%). Vital status was obtained in 160 patients (98%). Median follow-up was 6.9 years (range: 0.8 to 24.5 years). Perioperative mortality for all patients was 6%. Idiopathic CP had the best prognosis (7 year survival: 88%, 95% confidence interval [CI] 76% to 94%) followed by postsurgical (66%, 95% CI 52% to 78%) and postradiation CP (27%, 95% CI 9% to 58%). In bootstrap-validated proportional hazards analyses, predictors of poor survival were prior radiation, worse renal function, higher pulmonary artery pressure (PAP), abnormal left ventricular (LV) systolic function, lower serum sodium level, and older age. Pericardial calcification had no impact on survival. Conclusion: Long-term survival after pericardiectomy for CP is determined by the underlying etiology of constriction, LV systolic function, renal function, serum sodium, and PAP. Patients with postsurgical as well as postradiation CP have a survival inferior to patients with idiopathic CP. Perioperative Mortality is low. The relatively good survival after pericardiectomy in patients with idiopathic CP emphasizes the safety of pericardiectomy in this group.
Mesenchymal stem cells (MSC), also referred to as marrow stromal cells, maintain the capacity to differentiate into multiple mesenchymal lineages such as osteoblasts, chondrocytes, adipocytes, myoblasts, stromal, neural and endothelial cells. The use of autologous MSC has generated widespread interest due to their developing application in regenerative medicine and tissue engineering in orthopedic surgery. They have become an indispensable cell source for successful implementation in many bone reconstruction procedures. In addition to their multipotency and selfrenewal capacity, they are easily harvested, have demonstrated a homing mechanism and can be efficiently expanded in vitro, thus providing a safe and costefficient tissue replacement for patients with skeletal injury or disease. Little information is currently available concerning donor characteristics for tissue engineering growth of osseous tissue. This study examines the influences of such donor characteristics, including injury pattern, gender, age, and site of harvest on the quantity, quality and osteogenic differentiation of MSC. The goal is to evaluate whether certain patient groups are practically suitable for an ex vivo expansion and therapeutic reimplantation of MSC. The effect of injury pattern on the reservoir and proliferative capacity of MSC in human bone marrow is clearly demonstrated in this analysis. Age and gender were also shown to influence MSC number and proliferation, as in previous studies. A total of 53 participants (46 patients and 7 healthy volunteers ranging from 18 to 64 years of age), who were scheduled to undergo operative procedures on the pelvis, vertebrae, tibia or hip as well as cancellous bone autografts for reconstruction of various bone defects, were included in the study. Participants were divided into 4 groups for each gender: single fracture, multiple trauma, atrophic nonunion and healthy volunteers. A minimum of 6 ml bone marrow samples were aspirated intraoperatively and processed immediately according to protocol. Following cultivation and expansion for 14 days, the cells were then stained for the colony forming unit-fibroblast (CFU-F) assay and each culture flask was photographed, digitized and converted to an 8 bit grey level TIF-format. Using the digitized CFU-F assay, the mean colony number, mean colony area and mean cell number per microscopic field of view (cell density) could be determined. In addition, confirmation of MSC phenotype was established using fluorescent activated cell sorting (FACS). MSC potential for osteogenic differentiation was quantified by von Kossa, alkaline phosphatase and alizarin staining. Furthermore, serum from a total of 39 randomly chosen participants was collected and tested for hormone levels of 17β-estradiol, testosterone and prolactin as well as the cytokine interleukin-6. These analyses demonstrate several significant trauma-related modifications in MSC reservoir and proliferation, in both male and female patients. In multiple trauma patients, the highest MSC frequency was found, independent of gender and age. Proliferative capacity was also highest in male multiple trauma patients. In the case of atrophic nonunion, the lowest MSC reservoir was detected, independent of gender. Furthermore, MSC frequency in male patients was significantly higher than in female, although analyses of hormone and interleukin-6 levels provided no correlation. Agerelated changes in MSC reservoir could also be observed, whereas the proliferative capacity produced only a tendency toward decreasing values with increasing age. Concerning the site of cell harvest, MSC isolated from the proximal extremity of the tibia, greater trochanter and vertebral body did not proliferate sufficiently enough to be included in statistical analysis, supporting the use of the iliac crest for efficient expansion of MSC. This data suggests the interaction of yet to be identified processes in bone marrow in multiple trauma situations which stimulate the activation and mobilization of MSC. Moreover, in the case of atrophic nonunion, the concentration in bone marrow is depleted and the absence of systemic stimulation present in multiple trauma results in reduced activation of proliferative capacity. Such patients, with severe injury or atrophic nonunion, represent a group of patients with an especially acute necessity for effective and successful bone reconstruction. This data can be used to determine the applicability of MSC from various patient groups for osseous tissue replacement procedures. Especially in such medically challenging situations, further research is essential not only to delineate the factors involved in MSC regulation but also to develop methods to stimulate MSC expansion and proliferation.
Background The purpose of the trial was to evaluate the safety and performance of the new Protégé stent in the treatment of common and/or internal carotid artery stenoses. Methods The Protégé® GPS stent is a self-expanding Nitinol stent system. It is mounted on a 6 Fr 0.018” (6-9mm stent) or 7 Fr, 0.035” (10mm stent) over-the-wire-delivery system and includes a new stent release system which allows exact placement of the stent. Seventyseven patients were enrolled in the trial. Study patient assessments were conducted clinically and by duplex scan at baseline, peri-procedure, discharge, one and six months post procedure. Results Seventyseven lesions were treated. Thirtyone lesions were symptomatic, 46 lesions were asymptomatic. The procedure was technically successful in 76 (99%) lesions. The percentage of stenosis was reduced from 86 ± 7 % to 16 ± 8 %. One procedure failed because the embolic protection device could not be retrieved and the patient was sent to surgery. Within 30 days there were 4 (5.2%) Major Adverse Neurological Events (MANEs). Three of the MANEs were major strokes (3.9%), one a minor stroke. The fifth MANE occurred prior to the six month follow-up visit; this patient had a major stroke 75 days after the procedure and died 36 days later. One additional death occurred due to urosepsis. Conclusions The trial shows that the Protégé stent satisfies safety and performance criteria for the treatment of carotid artery stenosis. The complication rate was comparable to the incidence of these events in other recent carotid stent and endarterectomy studies.
Visual information is processed hierarchically in the human visual system. Early during processing basic features are analysed separately while at later stages of processing, they are integrated into a unified percept. By investigating a basic visual feature and following its integration at different levels of processing one can identify specific patterns. In certain visual impairments, these patterns can function defectively and their detailed study can clarify the cause of the visual deficit. Here we investigate orientation as a basic feature and use a property of the visual system called adaptation. Adaptation occurs as a decrease in the level of neural activity during repetitive presentation of the same stimulus. Psychophysical studies have shown that adaptation transfers interocularly, meaning that if only one eye is adapted the other eye shows also adaptation effects. Our aim was to investigate interocular transfer by means of functional magnetic resonance imaging (fMRI). Even though adaptation was demonstrated in the fMRI environment, the interocular transfer was never investigated in such a setup. First, we developed a method to measure interocular transfer of adaptation to gratings with fMRI. We then went further to test it in various groups of subjects. In normally sighted humans interocular transfer was present both in early (striate) as well as later visual areas (extrastriate). In subjects with impaired stereovision (with or without normal visual acuity) interocular transfer was absent in the investigated regions. Detailed analysis of the recorded differences between subjects with and subjects without stereovision was performed. The results of this analysis are presented in detail in this book. These results suggest that the neuronal mechanisms involved in the interocular transfer of pattern adaptation share, at least in part, the neural circuitry underlying binocular functions and stereopsis. We conclude that fMRI adaptation can be used for the assessment of cortical binocularity in humans with normal and impaired stereopsis. Further investigations are needed to address more subtle aspects of the lack of interocular transfer. Towards this purpose, through a fourth experiment we propose further directions that might shed more light on the issue of stereovision and its clinical implications. We show that carefully tuned variations in our experimental procedure might reveal other aspects of binocularity in the human visual system. We believe that the method we developed, apart from the interesting results shown here, has a high potential to be further used for other research questions. Following the above summarized ideas, the thesis comprises of three parts (chapters). The first chapter provides the main theoretical backgrounds of the visual system and of the MRI imaging technique, chapter two describes the experimental procedures while the results and their detailed discussion are detailed in chapter three.
Aims: The purpose of this study was to evaluate the feasibility and short-term efficacy of transcatheter paravalvular leak closure using different occlusion devices. Methods and Results: Twenty one patients underwent transcatheter closure of either aortic or mitral paravalvular leak from June 2002 to February 2006 using the Amplatzer PDA, ASD or VSD occluder. All patients had symptoms and signs of haemolysis and/or cardiac decompensation with dyspnoea. Implantation of a device was technically successful in twenty patients (95 %). Immediate residual leak was found in seventeen patients (85 %). Significant shunting persisted in nine cases during follow up (45 %). Permanent leaflet obstruction was observed in one patient. Severe complications during follow up led to early death in one patient and surgical intervention in three. A successful second catheter treatment was performed in another three patients. The event-free survival from reoperation, death and stroke at the end of the observation period was 80 %. Conclusion: Transcatheter closure of paravalvular leaks is a technically feasible, but demanding procedure. Residual leaks are common and may worsen pre-existing haemolysis. Due to the significant ongoing morbidity in this group of patients and the complexity of follow up individual patient results differ considerably. Nevertheless, it is possible to achieve some symptomatic relief, thus an interventional approach should be discussed as a potential treatment option for those patients with a limited defect and who are not deemed suitable for another operation.
End-stage renal disease has been denominated a vasculopathic state, owing to the accelerated arterial stiffening, which occurs in addition to and independent of atherosclerosis and bears an increased cardiovascular risk. The altered metabolic milieu in uraemia leads to an increased oxidative stress, heightened inflammatory burden, and an abnormal calcium-phosphate metabolism, which are thought to be responsible for the vascular changes. The pulse wave velocity (PWV) is a widely employed surrogate parameter of arteriosclerosis. The purpose of this study was to gain more insight into the pathogenesis of arterial stiffness, by investigating the influence of markers of oxidative stress, procoagulation, and inflammation, and of the calcium-phosphate product on the PWV. We conducted a cross-sectional study in 53 stable patients aged 59 ± 16 years, who had been on haemodialysis for at least 4 months (68 ± 48). Carotid-radial PWV was measured using a semi-automated device, Complior SP (Artech Medical, France). Advanced glycosylation end-products (AGE) and advanced oxidation protein products (AOPP), were quantified according to previously described methods. High sensitive CRP was measured using ELISA, whereas the other biochemical parameters, i.e. fibrinogen, albumin, calcium, phosphate, cholesterol, and triglycerides, were determined using routine methods. For statistical calculations we employed SPSS (Statistical Package of Social Science, 12.0, 2003). The correlations between PWV, as the dependent variable, and many dependent variables were assessed by means of multiple regression analysis, in which we controlled for the influence of the traditional cardiovascular risk factors and some of the patients’ medication (calcium-channel blockers and statins). PWV was found to be significantly correlated to serum CRP (p=0.003), LDLcholesterol (p<0.001), triglycerides (p<0.001), AGE (p=0.002), calcium (p<0.001), phosphate (p=0.001), and fibrinogen (p=0.020). Between PWV and dialysis duration (months) an interesting quadratic relationship (p=0.058) was noted. Against expectation, regression analysis showed a negative correlation between AOPP and PWV (p=0.001). We failed to confirm the correlation between PWV and age, systolic blood pressure, or heart rate. Among traditional cardiovascular risk factors only LDL-cholesterol was positively correlated to PWV. In this cross-sectional analysis we could put forward that PWV correlates positively and significantly with fibrinogen, CRP, AGEs, calcium, phosphate, and LDL-cholesterol in haemodialysis patients. It seems procoagulatory and proinflammatory pathways, oxidative stress, and the calcium-phosphate product exert a synergistic effect on disturbances of vascular architecture in ESRD patients.
The µ-opioid receptor is the primary target structure of most opioid analgesics and thus responsible for the predominant part of their wanted and unwanted effects. Carriers of the frequent genetic µ-opioid receptor variant N40D (allelic frequency 8.2 - 17 %), coded by the single nucleotide polymorphism A>G at position 118 of the µ-opioid receptor coding gene OPRM1 (OPRM1 118A>G SNP), suffer from a decreased opioid potency and from a higher need of opioid analgesics to reach adequate analgesia. The aim of the present work was to identify the mechanism by which the OPRM1 118A>G SNP decreases the opioid potency and to quantify its effects on the analgesic potency and therapeutic range of opioid analgesics.
To elucidate the consequences of the OPRM1 118A>G SNP for the effects of opioid analgesics, brain regions of healthy homozygous carriers of the OPRM1 118A>G SNP were identified by means of functional magnetic resonace imaging (fMRI), where the variant alters the response to opioid analgesics after painful stimulation. Afterwards, the µ-opioid receptor function was analyzed on a molecular level in post mortem samples of these brain regions. Finally, the consequences of the OPRM1 118A>G SNP for the analgesic and respiratory depressive effects of opioids were quantified in healthy carriers and non-carriers of OPRM1 118A>G SNP by means of experimental pain- and respiratory depression-models.
To identify pain processing brain regions, where the variant alters the response to opioid analgesics after painful stimulation, we investigated the effects of different alfentanil concentration levels (0, 25, 50 and 75 ng/ml) on pain-related brain activation achieved by short pulses (300 msec) of gaseous CO2 (66% v/v) delivered to the nasal mucosa using a 3.0 T magnetic head scanner in 16 non-carriers and nine homozygous carriers of the µ-opioid receptor gene variant OPRM1 118A>G. In brain regions associated with the processing of the sensory dimension of pain (pain intensity), such as the primary and secondary somatosensory cortices and the posterior insular cortex, the activation decreased linearly in relation to alfentanil concentrations, which was significantly less pronounced in OPRM1 118G carriers. In contrast, in brain regions known to process the affective dimension of pain (emotional dimension), such as the parahippocampal gyrus, amygdala and anterior insula, the pain-related activation disappeared already at the lowest alfentanil dose, without genotype differences.
Subsequently, we investigated the µ-opioid receptor-expression ([3H]-DAMGO saturation experiments, OPRM1 mRNA analysis by means of RT-PCR), the µ-opioid receptor affinity ([3H]-DAMGO saturation and competition experiments) and µ-opioid receptor signaling ([35S]- GTPγS binding experiments) in post mortem samples of the human SII-region, as a cortical projection region coding for pain intensity, and lateral thalamus, as an important region for nociceptive transmission. Samples of 22 non-carriers, 21 heterozygous and three homozygous carriers of OPRM1 118A>G SNP were included into the analysis. The receptor expression and receptor affinity of both brain regions did not differ between non-carriers and carriers of the variant N40D. In non-carriers, the µ-opioid receptors of the SII-region activated the receptor bound G-protein more efficiently than those of the thalamus (factor 1.55-2.27). This regional difference was missing in heterozygous (factor 0.78-1.66) and homozygous (factor 0.66-1.15) carriers of the N40D variant indicating a reduced receptor-G-protein-coupling in the SII-region.
Finally, the consequences of the alteration of µ-opioid receptor function in carriers and noncarriers of the genetic variant was investigated using pain- and respiratory depression-models. Therefore, 10 healthy non-carriers, four heterozygous and six homozygous carriers of the µ- opioid receptor variant N40D received an infusion of four different concentrations of alfentanil (0, 33.33, 66.66 and 100 ng/ml). At each concentration level, analgesia was assessed by means of electrically (5 Hz sinus 0 to 20 mA) and chemically (200 ms gaseous CO2 pulses applied to the nasal mucosa) induced pain, and respiratory depression was quantified by means of hypercapnic challenge according to Read and recording of the breathing frequency. The results showed that depending on the used pain model, both heterozygous and homozygous carriers of the variant N40D needed 2 – 4 times higher alfentanil concentrations to achieve the same analgesia as non-carriers. This increase seems to be at least for homozygous carriers unproblematic, because to reach a comparable respiratory depression as non-carriers, they needed 10-12 times higher alfentanil concentrations.
The results of this work demonstrate that the µ-opioid receptor variant N40D causes a regionally limited reduction of the signal transduction efficiency of µ-opioid receptors in brain regions involved in pain processing. Thus, the painful activation of sensory brain regions coding for pain intensity is not sufficiently suppressed by opioid analgesics in carriers of the variant N40D. Due to the insufficient suppression in hetero- and homozygous carriers of the variant N40D, the concentration of opioids has to be increased by a factor 2 - 4, in order to achieve the same analgesia as in non-carriers. At the same time, the respiratory depressive effects are decreased to a greater extent in homozygous carriers of the N40D variant as they need a 10 - 12 times higher opioid concentration to suffer from the same degree of respiratory depression as non-carriers. Due to the increased therapeutic range of opioid analgesics, an increase of the opioid dose seems to be harmless, at least for homozygous carriers of the N40D variant.
The removal of apoptotic cells (AC) can be regarded as an integral component of the program to terminate inflammation. Clearance of AC by professional phagocytes such as macrophages induces an anti-inflammatory phenotype in the latter ones. Anti-inflammatory or M2 polarization is also observed in macrophages infiltrating certain human tumors. These tumor-associated macrophages (TAM) contribute actively to tumor progression by promoting immune evasion, angiogenesis and tumor cell survival. The aim of my Ph.D. thesis was to approach the mechanisms as well as the characteristics of macrophage phenotype alterations induced by AC, and to elucidate a possible connection between tumor cell apoptosis and TAM generation. In the first part of my studies, I investigated the impact of AC on macrophage viability. I could show that macrophage survival against pro-apoptotic agents increased after the interaction with AC. Protection of macrophages against cell death required activation of phosphatidylinositol-3 kinase (PI3K), extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca2+ signaling, and correlated with Bcl-XL and Bcl-2 up-regulation as well as Ser136-Bad phosphorylation. Unexpectedly, neither phagocytosis nor binding of apoptotic debris to the phagocyte was necessary to induce protection. AC released the bioactive lipid sphingosine-1-phosphate (S1P), dependent on sphingosine kinase (SphK) 2, as a survival messenger. These data indicated an active role of AC in preventing cell destruction in their neighborhood. My next aim was to elucidate the mechanism of S1P production by AC. During cell death, SphK 2 was cleaved at its N-terminus by caspase-1. Thereupon, the truncated but enzymatically active fragment of SphK 2 was released from cells. This release was coupled to phosphatidylserine exposure, a hallmark of apoptosis and a crucial signal for the phagocyte/apoptotic cell interaction. Thus, I observed a link between common signaling events during apoptosis and the extracellular production of S1P, which is known to affect immune cell attraction and polarization as well as angiogenesis in cancer. In the next part of my studies, I asked for a correlation between tumor cell apoptosis and TAM polarization. During co-culture of human macrophages with human breast cancer carcinoma cells (MCF-7), the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-α; and interleukin (IL)-12-p70 production, but increased formation of IL-8 and IL-10. Alternative macrophage activation required tumor cell death, because a co-culture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. These phenotype alterations were also achieved with conditioned media from apoptotic tumor cells, which again argued for a soluble factor being involved. Knock-down of SphK2, but not SphK1, to attenuate S1P formation in MCF-7 cells, repressed the otherwise observed alternative macrophage polarization during co-culture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P was characterized by suppression of pro-inflammatory nuclear factor (NF)-κB DNA binding. These findings suggested that tumor cell apoptosis-derived S1P contributes to the macrophage polarization present in human tumors. To validate these in vitro data, I used an in vivo tumor model to clarify the relevance of SphK2 and S1P in tumor development. The growth of, as well as blood vessel infiltration into SphK2 knock-down MCF-7 (MCF-7-siSphK2) xenografts in nude mice was markedly decreased in comparison to control MCF-7 xenografts. In contrast, macrophage infiltration was similar or even more pronounced. These data provided a first hint for an in vivo role of SphK2-derived S1P in macrophage polarization associated with tumor promotion. In summary, these data indicate a new mechanism how AC themselves shape macrophage polarization, which results in the termination of inflammatory responses and macrophage survival. Furthermore, my studies present evidence that human tumors may utilize this mechanism to foster growth via increased angiogenesis.
Imatinib (GleevecTM; GlivecTM; formerly STI571), a specific inhibitor of Abl tyrosine kinase, is efficacious in treating Philadelphiachromosomepositive (Ph+) leukaemias such as chronic myeloid leukaemia (CML) and Ph+ acute lymphoblastic leukaemia (ALL) (Ottmann, Druker et al. 2002). Within a few years of its introduction to the clinic, Imatinib had dramatically altered the firstline therapy for CML, because it was found that most newly diagnosed CML patients in the chronic phase achieve durable responses when treated with Imatinib (Goldman and Melo 2003). However, a small percentage of these patients, as well as most advancedphase CML and Ph+ ALL patients, relapse on Imatinib therapy (Yokota, Kimura et al. 2006). Several mechanisms of refractoriness and relapse have been reported. These include point mutations within the Abl kinase domain, overexpression of BcrAbl mRNA (Hofmann, Jones et al. 2002), decreased intracellular drug levels mediated by Pglycoprotein (Pgp) (Hegedus, Orfi et al. 2002), and nonBcrAbl dependent mechanisms (activation of the SFKs) (Donato, Wu et al. 2003). In this research work, a possible means of overcoming resistance to Imatinib by the use of the specific dual Src/Abl kinase inhibitor AZD0530 has been investigated. The efficacy of AZD0530 in the treatment of Ph+ leukaemias, sensitive to or resistant to Imatinib, has been tested on cell lines, primary patient material and in vivo in transduction/transplantation mouse model of Imatinib sensitive or resistant BcrAbl dependent CML-like disease. Data with AZD0530 has been compared to cells treated with Imatinib. The potential of inhibiting both Src and Abl kinases while inducing growth arrest and apoptosis has been analysed. AZD0530 specifically inhibited the growth of CML and Ph+ ALL cells in a dosedependent manner, but has shown a marginal effect on Ph- ALL cells. Treatment of p185BcrAbl expressing Ba/F3 cells with AZD0530 has led to apoptosis induction and growth inhibition in these cells, while the untransformed Ba/F3 cells have remained unaffected. Resistance to Imatinib due to mutation in the Ba/F3MutY253F cells has been overcomed by this compound. The growth inhibitory effect of AZD0530 correlates with its induction of apoptosis. Combination of AZD0530 and Imatinib at low concentrations has shown an additive effect on the inhibition of proliferation of BV173 cells. The growth inhibition and apoptosis induction by AZD0530 have shown to be uncoupled to major changes in cell cycle. An exception is the CML blast crisis cell line BV173 which has shown a considerable G0/G1 arrest in the presence of AZD0530 and Imatinib as single agents. Immunoblotting of whole cell lysates from Imatinib or AZD0530 treated BV173, Ba/F3 expressing p185(BcrAbl) MutT253F cells and the WTSupB15 cells, for Src and BcrAbl clearly demonstrates that there is an ongoing transphosphorylation taking place between the SFKs and BcrAbl. This transphosphorylation synergizes and influences the aggressive nature of CML blast crisis and Ph+ ALL. Investigations have been carried out on downstream signaling events to determine how Src family members contribute to BcrAbl signaling. Specifically, Stat, Erk and PI3K/ Akt activation status have been characterised in Imatinib sensitive and resistant Ph+ cells. AZD0530 has significantly downregulated the activation of survival signaling pathways as shown by it’s inhibition of Stat5, Akt and Erk kinases in Ph+ cells, resistant or sensitive to Imatinib. The only exception to this has been the Imatinib resistant cell line RTSupB15, in which activated Akt kinase level has remained unaffected. AZD0530 has shown to be efficient in the treatment of cells isolated from three Ph+ leukaemic patients (resistant or sensitive to Imatinib), and has led to an induction of apoptosis. Equally, in the same patients, growth and survival pathways have been inhibited in vitro in the presence of AZD0530. An overall therapeutic effect of AZD0530 in vivo has been studied in mouse model of Imatinib sensitive and Imatinib resistant, BcrAbldependent desease. Mice with a BcrAbllike disease responded to Imatinib treatment but not to AZD0530. Using the CFU assay, an influence on the differentiation status of primary leukaemic blast stem cells have been tested. The in vivo studies as well as the CFU results have shown discrepancies to the effects of AZD0530 tested so far in this research work. These discrepancies have paralleled with the upregulation of BcrAbl in most AZD0530 treated cells. These are to be further analysed. These data elucidate the role of Src kinases in BcrAbl leukaemogenesis. Results gotten from this research work has shown that AZD0530 targets both Src and BcrAbl kinase activity and reduces the transforming potential of BcrAbl. It also shows that there is an ongoing transphosphorylation between SFKs and BcrAbl kinase. AZD0530 has proven effective in CML cell lines, Ph+ ALL cell lines and patient cells resistant to Imatinib. These have demonstrated that AZD0530 is a potential drug target which can be used to overcome Imatinib resistance.
Transcranial magnetic stimulation (TMS) is a non-invasive technique which can be used to study different intracortical excitatory and inhibitory neuronal circuits in the intact human being. In the primary motor cortex, there are essentially three different TMS measures of inhibitory neuronal circuits as determined by paired-pulse TMS: short-interval intracortical inhibition (SICI), long-interval intracortical inhibition (LICI) and interhemispheric inhibition (IHI). It was hypothesized that SICI is a GABAA receptor mediated inhibition (Ilic et al., 2002) whereas LICI and IHI are mediated by GABAB receptors (Daskalakis et al., 2002; McDonnell et al., 2006). Additionally, it was shown that these inhibitory circuits interact negatively, possible due to presynaptic GABAB receptor mediated inhibition (Sanger et al., 2001; Daskalakis et al., 2002). Which neuronal populations exactly underlie SICI, LICI and IHI, is not completely clear and by which mechanism these inhibitory circuits interact has never been tested pharmacologically so far. Thus, the effects of a single oral dose of Diazepam (DZP), a specific positive allosteric modulator at the GABAA receptor, and of Baclofen (BAC), a specific GABAB receptor agonist, on SICI, LICI and IHI as well as their interactions were tested here in a randomized, placebo controlled, double-blinded crossover study. SICI significantly increased after intake of DZP whereas BAC did not change SICI. Conversely, LICI significantly increased after intake of BAC but did not change after intake of DZP. IHI showed only a trend towards a decrease after intake of DZP but no change after intake of BAC. The interactions IHI-SICI, LICI-IHI and LICI-SICI were all negative at baseline. SICI and IHI were partially suppressed in the presence of IHI and LICI, respectively, and SICI in the presence of LICI was almost completely blocked. BAC did not change any of these interactions, whereas DZP significantly increased SICI in the presence of LICI. This study is the first to examine by means of pharmacological testing the complex interactions between different inhibitory circuits in the human motor cortex. The effects of DZP and BAC on SICI and LICI confirmed the notion that SICI is a GABAA receptor mediated intracortical inhibition whereas LICI depends on GABAB receptor mediated neurotransmission. The pharmacology of IHI at short interstimulus intervals of < 20 ms (12 ms in this study) remains still inconclusive and warrants further investigation. Findings further suggest that SICI, LICI and IHI represent three different inhibitory neuronal circuits which can be tested non-invasively by means of paired-pulse TMS. Furthermore, the data support the idea that the negative interactions IHI-SICI, LICI-IHI and LICI-SICI are most likely due to presynaptic GABAB receptor mediated autoinhibition.
Prostaglandin D2 (PGD2) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood and the data obtained so far are rather controversial. Here we investigated the effects of central PGD2 delivery and of systemic prostaglandin D synthase (PGDS) or cyclooxygenase (COX) inhibition on core body temperature (TC) and on prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Both PGE2 and PGD2 were detectable in CSF samples from control rats (6.2 ± 1.1 and 17.3 ± 3.1 pg/ml, respectively). Lipopolysaccharide (LPS) injection (50 μg i.p.) induced fever during the 5-hour observation period. Five hours after LPS injection, the levels of PGE2 and PGD2 were increased in the CSF about 90-fold (541.0 ± 47.5 pg/ml) and 5-fold (95.4 ± 23.1 pg/ml), respectively. Administration of PGD2 (50 - 500 ng) into the cisterna magna (i.c.m) evoked a delayed fever response in a dose-dependent manner that was accompanied by increased levels of PGE2 in the CSF. RT-PCR analyses revealed that the increased levels of PGE2 after PGD2 administration were not caused by up-regulation of COX-2 or microsomal prostaglandin E synthase 1 (mPGES-1) in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD2 considerably sustained the pyrogenic effects of i.c.m. administered PGE2. Pretreatment with a novel PGDS inhibitor, EDJ300520 (10 – 40 mg/kg p.o.), 1 h prior to the LPS injection impaired the LPS-induced increase of both PGD2 and PGE2 in the CSF and inhibited the fever response. In contrast, administration of EDJ300520 3 h after LPS injection did not ameliorate the LPS-induced fever. Accordingly, the concentration of PGE2 in the CSF was not decreased after EDJ300520 treatment. However, the CSF levels of PGD2 were reduced after administration of a high dose of EDJ300520 (40 mg/kg). We also investigated the effects of antipyretic drugs on the CSF levels of PGE2 and PGD2 during LPS-induced fever. Four antipyretic drugs with different mechanisms of action were used, including ibuprofen (5 - 20 mg/kg), celecoxib (10 - 50 mg/kg), SC560 5 - 20 mg/kg), and paracetamol (50 - 150 mg/kg). Each drug was used in three different doses and was orally administered 3 h after the LPS injection. All drugs were capable to attenuate the LPS-induced fever. The decrease of TC paralleled the reduction of PGE2 levels in the CSF. Of note, there was a tendency to reduced PGD2 levels in the CSF after treatment with the antipyretic drugs. However, only SC560 and the high dose of celecoxib (50 mg/kg) reduced the PGD2 levels significantly. In summary, our experiments underscore the pivotal role of PGE2 as the principal downstream mediator of fever. Moreover, we demonstrate that PGD2 is also involved in the mechanisms underlying fever. Our data suggest that PGD2 exerts an indirect pyrogenic effect by modulating the availability of PGE2 in the CSF. Additional studies are needed to explore the exact mechanism by
Spontaneous carotid artery dissection-associated medial changes in a selected autopsy population
(2006)
Spontaneous carotid artery dissection (SCAD) is a major cause of stroke in young adults, yet its pathogenesis remains unclear. Hereditary connective tissue diseases, hormonal influences, sympathomimetic drugs or upper respiratory tract infections may predispose to dissection. Mechanical stress or minimal trauma may also act as a trigger. Various lesions of the arterial wall have been described in association with SCAD, but no prospective autopsy study to evaluate the presence of these lesions in normal controls was found. We performed a histologic evaluation of the carotid bifurcation and the aortic arch in an autopsy series to establish a baseline anatomy of the bifurcation and to determine whether similar lesions could be observed. In seven of 54 (12.96%) selected cases we observed isolated changes closely resembling those described for medionecrosis, fibromuscular dysplasia, mucoid degeneration and elastinolysis; and in one case, prior carotid artery dissection and coiling with a patent false lumen. Generally, vascular microanatomy in the carotid bifurcation can be highly variable. Lesions similar to those associated with spontaneous dissection are present in controls and appear non-specific for spontaneous dissection. They can be explained as reactive changes of smooth muscle cells (SMC) and the vascular wall in response to various stressors. Recent advances in vascular physiology are discussed to illustrate the concept of SMC phenotypic modulation. Forensic pathology can provide a large control population for extensive vascular analyses and further the understanding of normal and pathological vascular wall changes to help elucidate spontaneous arterial dissection.
Background: False aneurysms at the puncture site develop in up to 8 % after catheter procedures. They can be treated surgically or by ultrasound guided manual compression. A new method is to inject thrombin into the aneurysm under ultrasound guidance. We evaluated safety and efficacy of this approach in a multicenter registry. Methods: In 595 consecutive patients (age: 31-94 years, median 70) a pseudoaneurysm (593 femoral arteries, 2 brachial arteries) was diagnosed 0 to 250 days (median 3 days) after a catheter procedure. The diameter of the aneurysm ranged from 0.5 x 0.5 x 0.5 (L x W x D) to 8x11x16 cm (median 2 x 2 x1.6 cm). 20 U to 4000 U of thrombin solution (median 400 U) were injected percutaneously into the aneurysm under ultrasound guidance. Results: The procedure was technically successful in 587/595 (99%) patients. The aneurysms were thrombosed after the first injection in 531 patients (89 %). Thirty-eight (6%) patients needed a second injection and 8 (1%) patients a third injection because residual flow in the aneurysm was visible at follow-up. In 4 additional patients (0.7%) the thrombosis of the aneurysms was delayed and occurred only after 24 hours to 7 days. 6 (1%) patients surgery was performed after successful closure of the aneurysm to remove the resulting haematoma. The overall technical success rate was 99% (587/595) and clinical success was achieved in 572/595 (96%) patients. Eight (1%) other patients underwent surgery due to thrombin injection failure. Complications occurred in 9 patients (1.5%): Intravascular thrombus formation (n=3), deep venous thrombosis (n=3), pulmonary embolism due to deep venous thrombosis (n=1), transient paresthesia in the leg during injection (n=3). Conclusion: Ultrasound guided thrombin injection is a safe, painless, effective and rapid alternative to treat false aneurysms. Complications and recurrent pseudoaneurysms are very rare. It has become the treatment of choice in our institution.
IL-12-related cytokines produced by dendritic cells are considered to be major inducers of adaptive immune system activation upon innate antigen-sensing. IL-23 specifically is currently being discussed to support the differentiation of potentially auto-aggressive Th17 cells. Prostaglandins as bystander cell products are known to modulate the translation of this process. While previous studies focused therefore on IL-12, ignoring the existence of new IL-12-related cytokines IL-23 and IL-27, this study analysed effects of prostaglandin E2, D2 and 15d-PGJ2 on the secretion pattern of these subunits in the murine immature Langerhans cell line XS52 and the murine immature myeloid dendritic cell line JawsII under TLR4 (LPS) and TLR9 (CpG) stimulation as well as effects of prostaglandins on the murine Th1 cell line IF12 in coculture and upon Con A treatment. In serial semi-quantitative RT-PCR of the IL-12 related cytokines of the XS52 cell line and the JawsII cell line, the p40 subunit was upregulated in both DC cell lines upon TLR-stimulation, the IL-23p19 subunit constantly expressed in XS52 and upregulated in JawsII upon TLR-stimulation, while the IL-27p28 subunit was only weekly expressed under additional stimulating aCD40 Ab treatment. IL-12p35 could only be detected in the immature myeloid cell line. The protein expression of the p40 subunit was measured in Western blot assays following SDS-PAGE under reducing conditions in XS52. The Western blot-based antibody specification allowed the establishment of a p40-specific ELISPOT assays, where overadditive upregulation of the number of LPS-stimulated spot forming XS52 cells was observed under stimulation with PGE2 while PGD2 depressed the number of LPS-stimulated cytokine secreting cells. Contrary IL-12p40 could not be detected in supernatants of the JawsII cell line. Both DC cell lines were further tested for differential response towards different TLR stimulation described as a defining feature of DC subsets. While subunit expression on transcription level did not differ, only LPS-treatment led to constant IL-12p40 expression in supernatants of XS52. CpG-treatment of XS52 cells led to constantly high IL-12p40 levels under additional aCD40 Ab treatment. In IFN-g ELISPOT assays, prostaglandin effects were further analysed in IF12 Th1 cells upon Con A treatment or alternatively upon treatment in a coculture model with the syngeneic cell line XS52 and the T lymphocyte-specific protein ovalbumin. While PGE2 depressed the amount of activated Th1, PGD2 showed no effect. In conclusion, a coculture model has been generated that allows the analysis of DC and TC interactions. The importance of prostaglandins as differential regulators in time- and tissue-dependence in inflammatory processes has been demonstrated. These results accord with recent observations of an upregulation of IL-23 secretion upon PGE2 treatment.
Molecular mechanism of intracellular signal transduction by the angiotensin-converting enzyme
(2007)
The angiotensin converting enzyme (ACE) is an important component of the renin-angiotensin system (RAS) and is crucially involved in the homeostasis of fluid and electrolyte balance and thus in the regulation of blood pressure. The zinc metallopeptidase is involved in the generation of angiotensin II, a potent vasoconstrictor and in the degradation of bradykinin, a potent vasodilator. It is worth noting that ACE more readily hydrolyzes bradykinin than it does angiotensin I thus culminating in the net physiological effect of the production of a vasoconstrictor and the decrease in the availability of a vasodilator. ACE inhibitors have become one of the most successful therapeutic approaches as a first line of therapy in hypertension, and are also widely used in treating heart failure, myocardial infarction, stroke, coronary artery disease and impaired left ventricular function. However, one unexpected clinically relevant finding related to ACE inhibitors is their ability to delay the onset of type II diabetes that was revealed by various large clinical trials. However, the mechanisms underlying these beneficial effects of ACE inhibitor therapy are currently unclear and cannot be explained by the prevention of angiotensin II formation or the attenuated degradation of bradykinin. Thus the potential beneficial effects attributed to ACE inhibitors may occur independent of reductions in blood pressure paving way for new and/or unknown mechanism. Our group has recently redefined ACE as a signal transduction molecule which upon binding to ACE inhibitor turns on a signalling cascade leading to phosphorylation of Ser1270 by CK2, activation of JNK and changes in gene expression in endothelial cells. However the mechanism by which ACE inhibitor initiates the signalling cascade was not clear. It was hypothesized that ACE, which is anchored to the membrane with a single transmembrane domain should dimerize prior to initiating further downstream signalling events in endothelial cells. Therefore, we sought to explore whether or not ACE forms dimers in endothelial cells and whether ACE dimerization is essential for the initiation of ACE signalling in endothelial cells. Using native gel electrophoresis, we found that ACE forms dimers in endothelial cells and that there is an increase in the dimer formation upon treatment of endothelial cells with ACE inhibitors. ACE homodimerization was also demonstrated using the split-ubiquitin system and chemical cross-linking experiments. ACE dimers are also formed in endothelial cells overexpressing the non-phosphorylatable ACE, wherein ACE signalling was abolished indicating that dimerization process is not influenced by the phosphorylation of the serine residue residing in the cytoplasmic tail. Monosaccharides like glucose, galactose and mannitol did not have any influence on ACE-inhibitor induced dimerization. Making use of different monoclonal antibodies directed to the epitopes of N-domain which harbours carbohydrate recognizing domain, also did not affect dimerization. However, inactivation of the C-domain active site by introducing mutation of the key histidine residues in HEMGH consensus sequences, which complexes the zinc ions, abolished enzyme dimerization both in the basal state and in response to ramiprilat. Mutation of the C-domain also resulted in the loss of ACE inhibitor-induced ACE signalling, that is we failed to observe ramiprilat-induced increase in the phosphorylation of the Ser1270 and the subsequent JNK activation. ACE-inhibitor induced dimerization precedes the phosphorylation of Ser1270 and activation of JNK. Thus the ACE-inhibitor induced dimerization via the C-domain of ACE represents the initial step in the ACE signalling pathway which involves the activation of JNK/c-Jun pathway and leading to the changes in the gene expression in endothelial cells. Our group previously identified ACE itself as well as cyclooxygenase-2 (COX-2) as two “ACE signalling-regulated” genes. To screen for additional genes regulated in a similar manner we used DNA microarray technology, to assess ramiprilat-induced changes in the endothelial cell gene expression. 21 genes were identified to be differentially regulated of which, 7 were upregulated and 14 were downregulated by ramiprilat. However, when screened at the protein level, we found no significant differences between the untreated control cells and those treated with ramiprilat. As several other cells and tissues possess a fully functional RAS we screened plasma samples from healthy volunteers as well as from patients with coronary artery disease for the proteins identified in the microarray. We observed that the cellular retinal binding protein-1 (CRBP-1) was detectable at low levels in plasma from patients and that ramipril markedly increased serum levels of this protein. Endothelial cells overexpressing CRBP-1 demonstrated increased RXRE and PPRE activity when stimulated with 9-cis retinoic acid and rosiglitazone respectively suggesting that CRBP-1 might affect gene expression via heterodimerization of PPAR elements with RXR elements by virtue of its function as a transport protein of retinoic acid. Studies aimed at determining the consequences of elevated CRBP-1 expression on endothelial cell homeostasis are ongoing. Although the RAS has been described in many other tissues apart from endothelial cells, ACE signalling has not yet been addressed in tissues such as monocytes/macrophages, which have an increased ACE expression in an atherosclerotic setting. We observed that upon stimulation of cultured ACE expressing monocytes with ramiprilat, JNK is activated suggesting the occurrence of ACE signalling in human monocytes. It is worth noting that ACE inhibitors delay the onset of type II diabetes in spite of moderate decrease in blood pressure. To further elucidate the mechanism underlying this effect, we found that ACE inhibitors increase the PPARgamma levels in the nuclear extracts of ACE expressing monocytes which were also reproduced in human endothelial cells overexpressing human somatic ACE. However, ramiprilat did not have any direct effect on the activity of a luciferase-coupled promoter containing several copies of the PPRE in human endothelial cells. These results contrasted with the actions of the PPARgamma agonist suggesting that ramiprilat enhances PPARgamma levels through an indirect mechanism. We next hypothesized that ramiprilat might increase the levels of 15-deoxy-D12,14-prostaglandin J2 (15dPGJ2) which is a natural ligand for PPARgamma via COX enzymes in monocytes. We observed that ramiprilat was able to decrease the diminution of COX-2 levels upto 48 hours of treatment but the levels of 15dPGJ2 were too low to be detected by ELISA. However ramiprilat enhanced the plasma levels of adiponectin, a downstream target of PPARgamma, which is a anti-atherogenic and anti-inflammatory adipokine, in patients with coronary artery disease. Though adiponectin is a PPARgamma-regulated gene, the observed increase in adiponectin might be attributed to the increase in RXR rather than via PPARgamma. Taken together, the results of this investigation have revealed that ACE inhibitors initiate ACE signalling by eliciting the dimerization of the enzyme, more specifically via its C-domain active centers. The ACE signalling cascade when activated leads to the enhanced expression of ACE, COX-2 and CRBP-1 which in turn favours the heterodimerization of PPARgamma with RXR and thus results in the increased expression of “PPARgamma regulated” genes such as adiponectin. The latter results provide a molecular basis for the observation that ACE inhibitors can delay the onset of type 2 diabetes in as much as it was possible to link ramipril with CRBP-1, RXR activity and the expression of adiponectin, an adipokine associated with improved insulin sensitivity. Further work is however required to elucidate the consequences of ACE inhibitors in monocytes and adipocytes as well as in intact animals.