Многомерно-временной операторный метод анализа и синтеза элементов САУ

Aims/hypothesis Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro. Methods Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA. Results A combination of the cytokines IL-1 beta, TNF-alpha and IFN-gamma induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets. Conclusions/interpretation These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines

Tissue Factor regulation, signaling and functions beyond coagulation with a focus on diabetes

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein best known for initiating the coagulation cascade upon binding of its ligand FVIIa. Apart from its physiological role in coagulation, TF and TF/FVIIa signaling has proved to be involved in diseases such as diabetes, cancer and cardiovascular diseases. Biological functions coupled to TF/FVIIa signaling include diet-induced obesity, apoptosis, angiogenesis and migration. Aim: The aim of this thesis was to investigate the role of TF/FVIIa in cells of importance in diabetes, to further investigate the mechanism behind TF/FVIIa anti-apoptotic signaling in cancer cells and lastly to examine the regulation of TF expression in monocytes by micro RNAs (miRNA). Results: In paper I we found that TF/FVIIa signaling augments cytokine-induced beta cell death and impairs glucose stimulated insulin secretion from human pancreatic islets. In paper II the relevance of TF/FVIIa in isolated human primary adipocytes was investigated. Adipocytes are a target cell for insulin and diabetics typically have increased lipolysis and impaired glucose uptake. No evidence was found for a role of TF/FVIIa in lipolysis or glucose uptake in adipocytes. However, adipocytes were found to express TF and FVII. The FVII produced was sufficient to initiate coagulation in the adipocytes. In paper III an anti-apoptotic TF/FVIIa induced signaling pathway in prostate and breast cancer cells was investigated in depth. Previous research has shown that TF/FVIIa signaling results in transactivation of insulin-like growth factor 1 receptor (IGF-1R) leading to subsequent protection from apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). The current results propose a mechanism where IGF-1R transactivation by TF/FVIIa is dependent on integrin β1 (ITGβ1) signaling. TF/FVIIa/ ITGβ1 signaling was found to result in phosphorylation of src and subsequent phosphorylation of caveolin 1 (Cav1). Once phosphorylated, the inhibitory effect of Cav1 on IGF-1R is cancelled, resulting in IGF-1R activation. In paper IV the role of miRNA regulation of TF expression in monocytic cells was investigated. The miRNA miR-223-3p was identified to be differentially expressed in U937 cells undergoing differentiation to a more monocyte-like phenotype and an anti-parallel correlation between TF and miR-223-3p expression in monocytes was proved. Hence, miR-223-3p regulates the inducible expression of TF in monocytes. Conclusions: The work in this thesis furthers the knowledge of molecular mechanisms behind TF regulation and TF/FVIIa signaling and some functional consequences as well as their biological relevance in diabetes.

Tissue Factor regulation, signaling and functions beyond coagulation with a focus on diabetes

Background: Tissue factor (TF) is a 47 kDa transmembrane glycoprotein best known for initiating the coagulation cascade upon binding of its ligand FVIIa. Apart from its physiological role in coagulation, TF and TF/FVIIa signaling has proved to be involved in diseases such as diabetes, cancer and cardiovascular diseases. Biological functions coupled to TF/FVIIa signaling include diet-induced obesity, apoptosis, angiogenesis and migration. Aim: The aim of this thesis was to investigate the role of TF/FVIIa in cells of importance in diabetes, to further investigate the mechanism behind TF/FVIIa anti-apoptotic signaling in cancer cells and lastly to examine the regulation of TF expression in monocytes by micro RNAs (miRNA). Results: In paper I we found that TF/FVIIa signaling augments cytokine-induced beta cell death and impairs glucose stimulated insulin secretion from human pancreatic islets. In paper II the relevance of TF/FVIIa in isolated human primary adipocytes was investigated. Adipocytes are a target cell for insulin and diabetics typically have increased lipolysis and impaired glucose uptake. No evidence was found for a role of TF/FVIIa in lipolysis or glucose uptake in adipocytes. However, adipocytes were found to express TF and FVII. The FVII produced was sufficient to initiate coagulation in the adipocytes. In paper III an anti-apoptotic TF/FVIIa induced signaling pathway in prostate and breast cancer cells was investigated in depth. Previous research has shown that TF/FVIIa signaling results in transactivation of insulin-like growth factor 1 receptor (IGF-1R) leading to subsequent protection from apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). The current results propose a mechanism where IGF-1R transactivation by TF/FVIIa is dependent on integrin β1 (ITGβ1) signaling. TF/FVIIa/ ITGβ1 signaling was found to result in phosphorylation of src and subsequent phosphorylation of caveolin 1 (Cav1). Once phosphorylated, the inhibitory effect of Cav1 on IGF-1R is cancelled, resulting in IGF-1R activation. In paper IV the role of miRNA regulation of TF expression in monocytic cells was investigated. The miRNA miR-223-3p was identified to be differentially expressed in U937 cells undergoing differentiation to a more monocyte-like phenotype and an anti-parallel correlation between TF and miR-223-3p expression in monocytes was proved. Hence, miR-223-3p regulates the inducible expression of TF in monocytes. Conclusions: The work in this thesis furthers the knowledge of molecular mechanisms behind TF regulation and TF/FVIIa signaling and some functional consequences as well as their biological relevance in diabetes.

Cationization increases brain distribution of an amyloid-beta protofibril selective F(ab')2 fragment

Antibodies and fragments thereof are, because of high selectivity for their targets, considered as potential therapeutics and biomarkers for several neurological disorders. However, due to their large molecular size, antibodies/fragments do not easily penetrate into the brain. The aim of the present study was to improve the brain distribution via adsorptive-mediated transcytosis of an amyloid-beta (A beta) protofibril selective F(ab')2 fragment (F(ab')2-h158). F(ab')2-h158 was cationized to different extents and the specific and unspecific binding was studied in vitro. Next, cationized F(ab')2-h158 was labelled with iodine-125 and its brain distribution and pharmacokinetics was studied in mice. Cationization did not alter the in vitro affinity to A beta protofibrils, but increased the unspecific binding somewhat. Ex vivo experiments revealed a doubling of brain concentrations compared with unmodified F(ab')2-h158 and in vivo imaging with single photon emission computed tomography (SPECT) showed that the cationized F(ab')2-h158, but not the unmodified F(ab')2-h158 could be visualized in the brain. To conclude, cationization is a means to increase brain concentrations of therapeutic antibodies or fragments and may facilitate the use of antibodies/fragments as imaging biomarkers in the brain

Tissue factor/factor VIIa signalling promotes cytokine-induced beta cell death and impairs glucose-stimulated insulin secretion from human pancreatic islets

Aims/hypothesis Patients diagnosed with type 1 or type 2 diabetes have elevated levels of coagulation factor VIIa (FVIIa) and its receptor tissue factor (TF) in their bloodstream. This may affect the fate of the beta cells. We aimed to study the effects of TF/FVIIa signalling on cytokine-induced beta cell death and islet function in vitro. Methods Human pancreatic islets and MIN-6 beta cells were used to study TF mRNA and protein expression using real-time PCR, immunoblotting and flow cytometry. The effects of TF/FVIIa on cytokine-induced beta cell death were studied in MIN-6 cells and human pancreatic islets using cell-death ELISA and propidium iodide and cleaved caspase-3 staining. Effects of TF/FVIIa on the phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) were investigated by immunoblotting. Glucose-stimulated insulin secretion (GSIS) from human islets was measured with an insulin ELISA. Results A combination of the cytokines IL-1 beta, TNF-alpha and IFN-gamma induced TF expression in human pancreatic islets and in beta cells. TF/FVIIa did not affect basal beta cell death but, independently of downstream coagulation activity, augmented beta cell death in response to cytokines. The effect of TF/FVIIa on cytokine-induced beta cell death was found to be dependent on the stress kinase JNK, since FVIIa addition potentiated cytokine-induced JNK activation and JNK inhibition abolished the effect of TF/FVIIa on cytokine-induced beta cell death. Moreover, TF/FVIIa signalling resulted in inhibition of GSIS from human pancreatic islets. Conclusions/interpretation These results indicate that TF/FVIIa signalling has a negative effect on beta cell function and promotes beta cell death in response to cytokines

Activation of β1 integrins and caveolin-1 by TF/FVIIa promotes IGF-1R signaling and cell survival

The tissue factor/coagulation factor VIIa (TF/FVIIa) complex induces transactivation of the IGF-1 receptor (IGF-1R) in a number of different cell types. The mechanism is largely unknown. The transactivation leads to protection from apoptosis and nuclear translocation of the IGF-1R. The aim of this study was to clarify the signaling pathway between TF and IGF-1R after FVIIa treatment with PC3 and DU145 prostate or MDA-MB-231 breast cancer cells as model systems. Protein interactions, levels, and phosphorylations were assessed by proximity ligation assay or flow cytometry in intact cells and by western blot on cell lysates. The transactivation of the IGF-1R was found dependent on TF/FVIIa-induced activation of β1-integrins. A series of experiments led to the conclusion that the caveolae protein caveolin-1 prevented IGF-1R activation in resting cells via its scaffolding domain. TF/FVIIa/β1-integrins terminated this inhibition by activation of Src family kinases and subsequent phosphorylation of caveolin-1 on tyrosine 14. This phosphorylation was not seen after treatment with PAR1 or PAR2 agonists. Consequently, the protective effect of FVIIa against apoptosis induced by the death receptor agonist TRAIL and the de novo synthesis of cyclin D1 induced by nuclear IGF-1R accumulation were both significantly reduced by down-regulation of β1-integrins or overexpression of the caveolin-1 scaffolding domain. In conclusion, we present a plausible mechanism for the interplay between TF and IGF-1R involving FVIIa, β1-integrins, Src family proteins, and caveolin-1. Our results increase the knowledge of diseases associated with TF and IGF-1R overexpression in general but specifically of TF-mediated signaling with focus on cell survival

Adipocytes express tissue factor and FVII and are procoagulant in a TF/FVIIa-dependent manner

Background: Tissue factor (TF) combined with its ligand FVII initiates blood coagulation and intracellular signaling. Obese and type 2 diabetic subjects have increased TF expression in their adipose tissue and an increased risk for thrombotic complications. Here we address the role of TF/FVII on adipocyte functions. Materials and methods: Subcutaneous fat was obtained by means of needle aspiration from healthy volunteers, and adipocytes were isolated after collagenase digestion. 3T3-L1 fibroblasts kept in culture were differentiated into adipocytes by addition of IBMX, dexamethasone, rosiglitazone, and insulin to the media. Proteins and mRNA were analyzed by western blot and RT-PCR. Coagulation activity was determined by a colorimetric FX-assay. Lipolysis was measured as free glycerol using a colorimetric method. Glucose uptake was evaluated by scintillation counting of D-[U-C-14] glucose. Results: In isolated human primary adipocytes we found expression of TF and FVII. TF expression was confirmed in 3T3-L1 adipocytes, and both cell types were found to be procoagulant in a TF/FVIIa-dependent manner. FXa was generated without FVIIa added to the coagulation assay, and active site-inhibited FVIIa blocked FXa formation, supporting our finding of FVII production by human primary adipocytes. There was no evidence for a role of TF in either lipolysis or glucose uptake in our experimental settings. Conclusion: Human primary adipocytes express active TF and FVII, and the TF/FVIIa complex formed on the adipocyte surface can activate substrate FX. Whether the TF/FVIIa complex conveys signaling pathways leading to biological functions and has any biological activity in adipocytes beyond coagulation remains to be elucidated