Molekularbiologische und zelltherapeutische Strategien zur Verbesserung der Vaskularisierung transplantierter Langerhans-Inseln

Die Transplantation von Langerhans-Inseln als therapeutischer Ansatz für Typ I Diabetes-Patienten stellt eine vielversprechende Alternative zur täglichen Insulinapplikation dar. Da die transplantierten Langerhans-Inseln allerdings bisher unzureichend revaskularisiert werden und daher ihre endokrine Funktion und ihr Überleben oft nicht dauerhaft garantiert sind, konnte sich diese Therapiemethode bisher nicht in der klinischen Routine durchsetzen. Entsprechend wird bis heute an der Entwicklung neuer Ansätze geforscht, um die Vaskularisierung transplantierter Langerhans-Inseln zu verbessern. Nerve/glial antigen (NG) 2 ist ein Proteoglykan, welches von Perizyten exprimiert wird und an der Regulation der Angiogenese beteiligt ist. Der Einfluss von NG2 auf die Inseltransplantation ist jedoch bis heute nicht bekannt. Daher wurde im ersten Studienabschnitt der vorliegenden Arbeit untersucht, ob NG2 die Revaskularisierung transplantierter Inseln fördert. Dazu wurden die Langerhans-Inseln von NG2+/+-Wildtyp- und NG2-/--Knockout-Mäusen zunächst mittels Immunhistochemie auf ihre zelluläre Zusammensetzung untersucht. Anschließend konnte die endokrine Funktion der isolierten Inseln mittels eines Insulin-Enzym-Immunassays analysiert werden. Zuletzt wurden die NG2+/+- und NG2-/--Inseln auf den quergestreiften Hautmuskel der Rückenhautkammer von NG2+/+-Mäusen transplantiert, um deren Revaskularisierung mittels intravitaler Fluoreszenzmikroskopie sowie immunhistochemischen Methoden untersuchen zu können. Im Rahmen dieser Analysen konnte gezeigt werden, dass sich NG2+/+- und NG2-/--Langerhans-Inseln weder in ihrer zellulären Zusammensetzung noch in ihrer Insulin-Sekretion unterscheiden. NG2-/--Inseln wiesen jedoch im Vergleich zu NG2+/+-Kontrollen eine signifikant reduzierte funktionelle Gefäßdichte sowie eine verringerte Anzahl CD31-positiver Blutgefäße an Tag 10 nach Transplantation auf. Folglich konnte gezeigt werden, dass der Verlust von NG2 die Revaskularisierung transplantierter Langerhans-Inseln beeinträchtigt. Dies verdeutlicht die Bedeutung dieses perizytären Oberflächenproteins für die Inseltransplantation. Im zweiten Studienabschnitt der vorliegenden Arbeit wurde untersucht, ob die Inkorporation von mikrovaskulären Fragmenten (MVF) in Inselorganoide deren Vaskularisierung verbessert. MVF werden aus Fettgewebe gewonnen und wurden bereits als potente Vaskularisierungseinheiten zur Prävaskularisierung von verschiedenen Geweben beschrieben. Bisher fanden sie allerdings noch keine Anwendung in der Inseltransplantation. Um prävaskularisierte Inselorganoide (PI+MVF) zu generieren, wurden Inselzellen und MVF aus C57BL/6N-Spendermäusen isoliert und über 5 Tage mithilfe der Liquid-Overlay-Technik co-kultiviert. Frisch isolierte Langerhans-Inseln, kultivierte Langerhans-Inseln und nicht prävaskularisierte Inselorganoide dienten als Kontrollen. Die Viabilität der Langerhans-Inseln und Inselorganoide wurde anhand von durchflusszytometrischen Analysen und der Verwendung von Spendermäusen, die einen endogenen Wasserstoffperoxid-Sensor exprimieren, bestimmt. Mithilfe von immunhistochemischen Untersuchungen und Western Blot-Analysen wurde die Interaktion zwischen den β- und Endothelzellen innerhalb der PI+MVF verifiziert. Die angiogene Aktivität der PI+MVF wurde in vivo unter Verwendung des murinen Rückenhautkammer-Modells und der repetitiven intravitalen Fluoreszenzmikroskopie sowie immunhistochemischer Methoden analysiert. Abschließend wurden PI+MVF in diabetische Empfängermäuse transplantiert, um die endokrine Funktion der Inselorganoide in vivo nachzuweisen. Im Rahmen dieser Analysen konnte gezeigt werden, dass die parakrine Signalübertragung zwischen β-Zellen und MVF die Viabilität und angiogene Aktivität der PI+MVF verbessert. In vivo wiesen die PI+MVF innerhalb der ersten Tage nach Transplantation eine beschleunigte Vaskularisierung auf, was anhand einer gesteigerten funktionellen Gefäßdichte und einem erhöhten Anteil an CD31-positiven Endothelzellen innerhalb der PI+MVF gezeigt werden konnte. Zudem induzierte die Transplantation von 250 Inselorganoiden in diabetische Mäuse innerhalb von 4 Tagen physiologische Blutglukosespiegel, während die identische Zahl frisch isolierter Langerhans-Inseln über den gesamten Beobachtungszeitraum von 28 Tagen nicht zu einer Normoglykämie führte. Diese Ergebnisse zeigen, dass die Inkorporation von MVF die Vaskularisierung von PI+MVF deutlich verbessert und die Wiederherstellung einer Normoglykämie in diabetischen Mäusen beschleunigt. Der hier vorgestellte Prävaskularisierungsansatz könnte damit eine vielversprechende Strategie sein, um in Zukunft die Erfolgsraten der klinischen Inselzelltransplantation zu erhöhen.Pancreatic islet transplantation is a promising therapeutic approach for the treatment of type 1 diabetic patients. However, this method is not yet established in clinical routine, because it is still limited by failed islet survival and endocrine function due to insufficient islet vascularization. To overcome this problem, current research focuses on strategies to improve the vascularization of transplanted islets. Nerve/glial antigen (NG) 2 is a proteoglycan, which is expressed on pericytes and a crucial regulator of angiogenesis. However, the influence of this proteoglycan on islet transplantation is still unknown. Accordingly, the aim of the first study section of the present thesis was to clarify whether NG2 promotes the revascularization of transplanted pancreatic islets. For this purpose, islets were isolated from NG2+/+ wild-type and NG2-/- knockout mice and analyzed by immunohistochemistry to examine their cellular composition. Their endocrine function was assessed by an insulin-enzyme-linked immunosorbent assay. Moreover, NG2+/+ and NG2-/- islets were transplanted into the dorsal skinfold chamber of NG2+/+ mice to study their revascularization by intravital fluorescence microscopy and immunohistochemistry. It could be shown that NG2+/+ and NG2-/- islets do neither differ in their cellular composition nor in their insulin secretion. However, NG2-/- islets exhibited a significantly reduced functional microvessel density and a lower number of CD31-positive blood vessels on day 10 after transplantation when compared to NG2+/+ controls. These results demonstrate that the loss of NG2 impairs the revascularization of transplanted islets, indicating the importance of this surface protein for successful islet transplantation. In the second study section of the present thesis, it was examined whether the incorporation of microvascular fragments (MVF) in islet organoids improves their vascularization after transplantation. MVF can be isolated from adipose tissue and have already been described as potent vascularization units for the prevascularization of various tissues. However, their impact on the engraftment of transplanted islets has not been analyzed, yet. For the generation of prevascularized islet organoids (PI+MVF), islet cells and MVF were isolated from C57BL/6N donor mice and co-cultured for 5 days by means of the liquid overlay technique. Freshly isolated islets, cultured islets and non-prevascularized islet organoids served as controls. The islets’ and islet organoids’ viability was determined by flow cytometric analysis and donor mice expressing an endogenous hydrogen peroxide sensor. Immunohistochemical studies and Western blot analysis were used to investigate the interaction between β-cells and endothelial cells within PI+MVF. The angiogenic potential of PI+MVF was determined by means of the mouse dorsal skinfold chamber model in combination with repetitive intravital fluorescence microscopy and immunohistochemical methods. Finally, PI+MVF were transplanted into diabetic recipient mice to verify their endocrine function in vivo. It could be shown that the paracrine signal transduction between β-cells and MVF improves the viability and angiogenic activity of PI+MVF. In vivo, PI+MVF exhibited an accelerated vascularization, as demonstrated by an increased functional microvessel density during the entire observation period and an increased fraction of endothelial cells 14 days after transplantation. Moreover, the transplantation of 250 PI+MVF into diabetic mice resulted in physiological blood glucose levels within 4 days, while the identical number of freshly isolated islets did not restore normoglycemia during the entire observation period of 28 days. Taken together, these results demonstrate that the incorporation of MVF significantly improves the vascularization of transplanted PI+MVF and accelerates the restoration of normoglycemia in diabetic mice. Thus, this prevascularization approach may be a promising strategy to increase the success of clinical islet cell transplantation in the future

Regulatory Mechanisms of Somatostatin Expression

Somatostatin is a peptide hormone, which most commonly is produced by endocrine cells and the central nervous system. In mammals, somatostatin originates from pre-prosomatostatin and is processed to a shorter form, i.e., somatostatin-14, and a longer form, i.e., somatostatin-28. The two peptides repress growth hormone secretion and are involved in the regulation of glucagon and insulin synthesis in the pancreas. In recent years, the processing and secretion of somatostatin have been studied intensively. However, little attention has been paid to the regulatory mechanisms that control its expression. This review provides an up-to-date overview of these mechanisms. In particular, it focuses on the role of enhancers and silencers within the promoter region as well as on the binding of modulatory transcription factors to these elements. Moreover, it addresses extracellular factors, which trigger key signaling pathways, leading to an enhanced somatostatin expression in health and disease

Protein Kinase CK2—A Putative Target for the Therapy of Diabetes Mellitus?

Since diabetes is a global epidemic, the development of novel therapeutic strategies for the treatment of this disease is of major clinical interest. Diabetes is differentiated in two types: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM arises from an autoimmune destruction of insulin-producing β-cells whereas T2DM is characterized by an insulin resistance, an impaired insulin reaction of the target cells, and/or dysregulated insulin secretion. In the past, a growing number of studies have reported on the important role of the protein kinase CK2 in the regulation of the survival and endocrine function of pancreatic β-cells. In fact, inhibition of CK2 is capable of reducing cytokine-induced loss of β-cells and increases insulin expression as well as secretion by various pathways that are regulated by reversible phosphorylation of proteins. Moreover, CK2 inhibition modulates pathways that are involved in the development of diabetes and prevents signal transduction, leading to late complications such as diabetic retinopathy. Hence, targeting CK2 may represent a novel therapeutic strategy for the treatment of diabetes

The Marine-Derived Triterpenoid Frondoside A Inhibits Thrombus Formation

Background: The marine-derived triterpenoid frondoside A inhibits the phosphatidylinositol-3-kinase (PI3K) pathway in cancer cells. Because this pathway is also crucially involved in platelet activation, we studied the effect of frondoside A on thrombus formation. Methods: Frondoside A effects on platelet viability, surface adhesion molecule expression, and intracellular signaling were analyzed by flow cytometry and Western blot. The effect of frondoside A was analyzed by photochemically induced thrombus formation in the mouse dorsal skinfold chamber model and by tail vein bleeding. Results: Concentrations of up to 15 µM frondoside A did not affect the viability of platelets, but reduced their surface expression of P-selectin (CD62P) and the activation of glycoprotein (GP)IIb/IIIa after agonist stimulation. Additional mechanistic analyses revealed that this was mediated by downregulation of PI3K-dependent Akt and extracellular-stimuli-responsive kinase (ERK) phosphorylation. Frondoside A significantly prolonged the complete vessel occlusion time in the mouse dorsal skinfold chamber model of photochemically induced thrombus formation and also the tail vein bleeding time when compared to vehicle-treated controls. Conclusion: Our findings demonstrated that frondoside A inhibits agonist-induced CD62P expression and activation of GPIIb/IIIa. Moreover, frondoside A suppresses thrombus formation. Therefore, this marine-derived triterpenoid may serve as a lead compound for the development of novel antithrombotic drugs

Protein Kinase CK2 Controls CaV2.1-Dependent Calcium Currents and Insulin Release in Pancreatic β-cells

The regulation of insulin biosynthesis and secretion in pancreatic β-cells is essential for glucose homeostasis in humans. Previous findings point to the highly conserved, ubiquitously expressed serine/threonine kinase CK2 as having a negative regulatory impact on this regulation. In the cell culture model of rat pancreatic β-cells INS-1, insulin secretion is enhanced after CK2 inhibition. This enhancement is preceded by a rise in the cytosolic Ca2+ concentration. Here, we identified the serine residues S2362 and S2364 of the voltage-dependent calcium channel CaV2.1 as targets of CK2 phosphorylation. Furthermore, co-immunoprecipitation experiments revealed that CaV2.1 binds to CK2 in vitro and in vivo. CaV2.1 knockdown experiments showed that the increase in the intracellular Ca2+ concentration, followed by an enhanced insulin secretion upon CK2 inhibition, is due to a Ca2+ influx through CaV2.1 channels. In summary, our results point to a modulating role of CK2 in the CaV2.1-mediated exocytosis of insulin

Erythropoietin exposure of isolated pancreatic islets accelerates their revascularization after transplantation

Aims The exposure of isolated pancreatic islets to pro-angiogenic factors prior to their transplantation represents a promising strategy to accelerate the revascularization of the grafts. It has been shown that erythropoietin (EPO), a glycoprotein regulating erythropoiesis, also induces angiogenesis. Therefore, we hypothesized that EPO exposure of isolated islets improves their posttransplant revascularization. Methods Flow cytometric, immunohistochemical and quantitative real-time (qRT)-PCR analyses were performed to study the effect of EPO on the viability, cellular composition and gene expression of isolated islets. Moreover, islets expressing a mitochondrial or cytosolic H2O2 sensor were used to determine reactive oxygen species (ROS) levels. The dorsal skinfold chamber model in combination with intravital fluorescence microscopy was used to analyze the revascularization of transplanted islets. Results We found that the exposure of isolated islets to EPO (3 units/mL) for 24 h does not affect the viability and the production of ROS when compared to vehicle-treated and freshly isolated islets. However, the exposure of islets to EPO increased the number of CD31-positive cells and enhanced the gene expression of insulin and vascular endothelial growth factor (VEGF)-A. The revascularization of the EPO-cultivated islets was accelerated within the initial phase after transplantation when compared to both controls. Conclusion These findings indicate that the exposure of isolated islets to EPO may be a promising approach to improve clinical islet transplantation

Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8

Angiogenesis crucially contributes to various diseases, such as cancer and diabetic retinopathy. Hence, anti-angiogenic therapy is considered as a powerful strategy against these diseases. Previous studies reported that the acyclic monoterpene linalool exhibits anticancer, anti-inflammatory and anti-oxidative activity. However, the effects of linalool on angiogenesis still remain elusive. Therefore, we investigated the action of (3R)-(−)-linalool, a main enantiomer of linalool, on the angiogenic activity of human dermal microvascular endothelial cells (HDMECs) by a panel of angiogenesis assays. Non-cytotoxic doses of linalool significantly inhibited HDMEC proliferation, migration, tube formation and spheroid sprouting. Linalool also suppressed the vascular sprouting from rat aortic rings. In addition, Matrigel plugs containing linalool exhibited a significantly reduced microvessel density 7 days after implantation into BALB/c mice. Mechanistic analyses revealed that linalool promotes the phosphorylation of extracellular signal-regulated kinase (ERK), downregulates the intracellular level of adenosine triphosphate (ATP) and activates the transient receptor potential cation channel subfamily M (melastatin) member (TRPM)8 in HDMECs. Inhibition of ERK signaling, supplementation of ATP and blockade of TRPM8 significantly counteracted linalool-suppressed HDMEC spheroid sprouting. Moreover, ATP supplementation completely reversed linalool-induced ERK phosphorylation. In addition, linalool-induced ERK phosphorylation inhibited the expression of bone morphogenetic protein (BMP)-2 and linalool-induced TRPM8 activation caused the inhibition of β1 integrin/focal adhesion kinase (FAK) signaling. These findings indicate an anti-angiogenic effect of linalool, which is mediated by downregulating intracellular ATP levels and activating TRPM8

Darbepoetin-α increases the blood volume flow in transplanted pancreatic islets in mice

Aims The minimal-invasive transplantation of pancreatic islets is a promising approach to treat diabetes mellitus type 1. However, islet transplantation is still hampered by the insufficient process of graft revascularization, leading to a poor clinical outcome. Accordingly, the identification of novel compounds, which accelerate and improve the revascularization of transplanted islets, is of great clinical interest. Previous studies have shown that darbepoetin (DPO)-α, a long lasting analogue of erythropoietin, is capable of promoting angiogenesis. Hence, we investigated in this study whether DPO improves the revascularization of transplanted islets. Methods Islets were isolated from green fluorescent protein-positive FVB/N donor mice and transplanted into dorsal skinfold chambers of FVB/N wild-type animals, which were treated with DPO low dose (2.5 µg/kg), DPO high dose (10 µg/kg) or vehicle (control). The revascularization was assessed by repetitive intravital fluorescence microscopy over an observation period of 14 days. Subsequently, the cellular composition of the grafts was analyzed by immunohistochemistry. Results The present study shows that neither low- nor high-dose DPO treatment accelerates the revascularization of free pancreatic islet grafts. However, high-dose DPO treatment increased the blood volume flow of the transplanted islet. Conclusions These findings demonstrated that DPO treatment does not affect the revascularization of transplanted islets. However, the glycoprotein increases the blood volume flow of the grafts, which results in an improved microvascular function and may facilitate successful transplantation

Early Cytokine-Induced Transient NOX2 Activity Is ER Stress-Dependent and Impacts β-Cell Function and Survival

In type 1 diabetes (T1D) development, proinflammatory cytokines (PIC) released by immune cells lead to increased reactive oxygen species (ROS) production in β-cells. Nonetheless, the temporality of the events triggered and the role of different ROS sources remain unclear. Isolated islets from C57BL/6J wild-type (WT), NOX1 KO and NOX2 KO mice were exposed to a PIC combination. We show that cytokines increase O2 •− production after 2 h in WT and NOX1 KO but not in NOX2 KO islets. Using transgenic mice constitutively expressing a genetically encoded compartment specific H2O2 sensor, we show, for the first time, a transient increase of cytosolic/nuclear H2O2 in islet cells between 4 and 5 h during cytokine exposure. The H2O2 increase coincides with the intracellular NAD(P)H decrease and is absent in NOX2 KO islets. NOX2 KO confers better glucose tolerance and protects against cytokine-induced islet secretory dysfunction and death. However, NOX2 absence does not counteract the cytokine effects in ER Ca2+ depletion, Store-Operated Calcium Entry (SOCE) increase and ER stress. Instead, the activation of ER stress precedes H2O2 production. As early NOX2-driven ROS production impacts β-cells’ function and survival during insulitis, NOX2 might be a potential target for designing therapies against early β-cell dysfunction in the context of T1D onset

Erythropoietin accelerates the revascularization of transplanted pancreatic islets

Background and Purpose Pancreatic islet transplantation is a promising therapeutic approach for Type 1 diabetes. A major prerequisite for the survival of grafted islets is a rapid revascularization after transplantation. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to promote angiogenesis. Therefore, we investigated in this study whether EPO improves the revascularization of transplanted islets. Experimental Approach Islets from FVB/N mice were transplanted into dorsal skinfold chambers of recipient animals, which were daily treated with an intraperitoneal injection of EPO (500 IU·kg−1) or vehicle (control) throughout an observation period of 14 days. In a second set of experiments, animals were only pretreated with EPO over a 6‐day period prior to islet transplantation. The revascularization of the grafts was assessed by repetitive intravital fluorescence microscopy and immunohistochemistry. In addition, a streptozotocin‐induced diabetic mouse model was used to study the effect of EPO‐pretreatment on the endocrine function of the grafts. Key Results EPO treatment slightly accelerated the revascularization of the islet grafts. This effect was markedly more pronounced in EPO‐pretreated animals, resulting in significantly higher numbers of engrafted islets and an improved perfusion of endocrine tissue without affecting systemic haematocrit levels when compared with controls. Moreover, EPO‐pretreatment significantly accelerated the recovery of normoglycaemia in diabetic mice after islet transplantation. Conclusion and Implications These findings demonstrate that, particularly, short‐term EPO‐pretreatment represents a promising therapeutic approach to improve the outcome of islet transplantation, without an increased risk of thromboembolic events