Maria Grandoch

The role of Epac proteins, novel cAMP mediators, in the regulation of immune, lung and neuronal function

Chronic degenerative inflammatory diseases, such as chronic obstructive pulmonary disease and Alzheimers dementia, afflict millions of people around the world, causing death and debilitation. Despite the global impact of these diseases, there have been few innovative breakthroughs into their cause, treatment or cure. As with many debilitating disorders, chronic degenerative inflammatory diseases may be associated with defective or dysfunctional responses to second messengers, such as cyclic adenosinemonophosphate (cAMP). The identification of the cAMP‐activated guanine nucleotide exchange factors for Ras‐like GTPases, Epac1 (also known as cAMP‐GEF‐I) and Epac2 (also known as cAMP‐GEF‐II), profoundly altered the prevailing assumptions concerning cAMP signalling, which until then had been solely associated with protein kinase A (PKA). Studies of the molecular mechanisms of Epac‐related signalling have demonstrated that these novel cAMP sensors regulate many physiological processes either alone and/or in concert with PKA. These include calcium handling, cardiac and smooth muscle contraction, learning and memory, cell proliferation and differentiation, apoptosis, and inflammation. The diverse signalling properties of cAMP might be explained by spatio‐temporal compartmentalization, as well as A‐kinase anchoring proteins, which seem to coordinate Epac signalling networks. Future research should focus on the Epac‐regulated dynamics of cAMP, and, hopefully, the development of compounds that specifically interfere with the Epac signalling system in order to determine the precise significance of Epac proteins in chronic degenerative inflammatory disorders.

Alternatively spliced human tissue factor (asHTF) is not pro-coagulant

It has been proposed that alternatively-spliced human tissue factor (asHTF) is pro-coagulant. We have evaluated the function of asHTF in a mammalian expression system. Full-length human tissue factor (HTF) and asHTF were cloned from smooth muscle cells and over-expressed in HEK293 cells. As expected, a marked pro-coagulant activity (FX activation, thrombin generation) was observed on the surface, in lysates, and on microparticles from HTF transfected cells. In contrast, no pro-coagulant activity of asHTF was observed.

Collagen Fragments Inhibit Hyaluronan Synthesis in Skin Fibroblasts in Response to Ultraviolet B (UVB): NEW INSIGHTS INTO MECHANISMS OF MATRIX REMODELING*

UVB irradiation causes characteristic features of skin aging including remodeling of the dermal extracellular matrix. A key feature during this process is the up-regulation of matrix metalloproteinases and cleavage of collagen. Hyaluronic acid (HA), a major component of the dermal matrix, decreases after chronic UVB exposure. However, the factors that govern the decline of HA synthesis during the course of actinic aging are largely unknown. The aim of the present study was to explore whether collagen degradation causes inhibition of HA synthesis in human skin fibroblasts. After treatment of fibroblasts with collagen fragments (CF) in vitro, resolution of the actin cytoskeleton and inhibition of HA secretion occurred because of specific down-regulation of hyaluronan synthase 2 (HAS2) expression. The αvβ3-agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to αvβ3 abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by αvβ3-blocking antibodies. The key role of ERK signaling was shown by reduced nuclear accumulation of phosphoERK and of ELK-1 phosphorylation in response to CF. In addition, the ERK inhibitor PD98059 reduced HAS2 expression. Also, UVB irradiation of fibroblasts caused down-regulation of HAS2, which was sensitive to matrix metalloproteinase inhibitors and to αvβ3-blocking antibodies. In conclusion, these data suggest that CF activate αvβ3-integrins and in turn inhibit Rho kinase (ROCK) signaling and nuclear translocation of phosphoERK, resulting in reduced HAS2 expression. Therefore, a novel mechanism is presented how proteolytic collagen cleavage may inhibit HA synthesis in dermal fibroblasts during extrinsic skin aging.

Blood Platelets in the Progression of Alzheimer's Disease

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.

Natural Antisense Transcript for Hyaluronan Synthase 2 (HAS2-AS1) Induces Transcription of HAS2 via Protein O-GlcNAcylation

Background: Intracellular proteins glycosylation with O-GlcNAc is able to influence cell microenvironment. Results: O-GlcNAcylation increases hyaluronan synthase 2 (HAS2) transcription via its natural antisense transcript HAS2-AS1. Conclusion: A novel mechanism to regulate hyaluronan synthesis via long non-coding RNA is described. Significance: This finding highlights a new target to regulate HA synthesis, critical in many pathophysiological processes. Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications.

Preliminary evidence for a matrix metalloproteinase-2 (MMP-2)-dependent shedding of soluble CD40 ligand (sCD40L) from activated platelets

Platelets are the major source of soluble CD40 ligand (sCD40L) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets to release sCD40L. However, the enzyme involved in sCD40L shedding has not been identified yet. Using a panel of pharmacological inhibitors of serine, cysteine, aspartate, or metalloproteinases, preliminary evidence is presented for the hypothesis that matrix metalloproteinase-2 (MMP-2) might be the protease, primarily responsible for CD40L cleavage from platelet surface.

Noninvasive Imaging of Early Venous Thrombosis by 19F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions

Background— Noninvasive detection of deep venous thrombi and subsequent pulmonary thromboembolism is a serious medical challenge, since both incidences are difficult to identify by conventional ultrasound techniques. Methods and Results— Here, we report a novel technique for the sensitive and specific identification of developing thrombi using background-free 19F magnetic resonance imaging, together with &agr;2-antiplasmin peptide (&agr;2AP)–targeted perfluorocarbon nanoemulsions (PFCs) as contrast agent, which is cross-linked to fibrin by active factor XIII. Ligand functionality was ensured by mild coupling conditions using the sterol-based postinsertion technique. Developing thrombi with a diameter <0.8 mm could be visualized unequivocally in the murine inferior vena cava as hot spots in vivo by simultaneous acquisition of anatomic matching 1H and 19F magnetic resonance images at 9.4 T with both excellent signal-to-noise and contrast-to-noise ratios (71±22 and 17±5, respectively). Furthermore, &agr;2AP-PFCs could be successfully applied for the diagnosis of experimentally induced pulmonary thromboembolism. In line with the reported half-life of factor XIIIa, application of &agr;2AP-PFCs >60 minutes after thrombus induction no longer resulted in detectable 19F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, &agr;2AP-PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. Conclusions— Our results demonstrate that 1H/19F magnetic resonance imaging, together with &agr;2AP-PFCs, is a sensitive, noninvasive technique for the diagnosis of acute deep venous thrombi and pulmonary thromboemboli. Furthermore, ligand coupling by the sterol-based postinsertion technique represents a unique platform for the specific targeting of PFCs for in vivo 19F magnetic resonance imaging.

B cell receptor-induced growth arrest and apoptosis in WEHI-231 immature B lymphoma cells involve cyclic AMP and Epac proteins

Signaling by the B cell antigen receptor (BCR) is essential for B lymphocyte homeostasis and immune function. In immature B cells, ligation of the BCR promotes growth arrest and apoptosis, and BCR-driven balancing between pro-apoptotic extracellular signal-regulated kinase 1 and 2 (ERK1/2) and anti-apoptotic phosphoinositide 3-kinase-dependent Akt seems to define the final cellular apoptotic response. Dysfunction of these late BCR signaling events can lead to the development of immunological diseases. Here we report on novel cyclic AMP-dependent mechanisms of BCR-induced growth arrest and apoptosis in the immature B lymphoma cell line WEHI-231. BCR signaling to ERK1/2 and Akt requires cyclic AMP-regulated Epac, the latter acting as a guanine nucleotide exchange factor for Rap1 and H-Ras independent of protein kinase A. Importantly, activation of endogenously expressed Epac by a specific cyclic AMP analog enhanced the induction of growth arrest (reduced DNA synthesis) and apoptosis (nuclear condensation, annexin V binding, caspase-3 cleavage and poly-ADP-ribose polymerase processing) by the BCR. Our data indicate that cyclic AMP-dependent Epac signals to ERK1/2 and Akt upon activation of Rap1 and H-Ras, and is involved in BCR-induced growth arrest and apoptosis in WEHI-231 cells.

Methods to evaluate the pharmacology of oral antiplatelet drugs.

Principally, there are two reasons why the pharmacological response to antiplatelet drugs should be measured: on the one hand, an insufficient inhibition of platelet function may result in atherothrombotic complications; on the other hand, an excessive inhibition of platelet function may lead to bleeding complications. The clinical importance to measure the effects of antiplatelet drugs is demonstrated by increasingly growing evidence for an association of resistance to antiplatelet drugs with thromboembolic events. It is often claimed that there is no generally accepted definition of “resistance” and, instead, there is an ongoing semantic discussion about the correct term to be used to describe this phenomenon. From the pharmacological point of view, there is only one acceptable definition of “resistance” to antiplatelet drugs: the term “resistance” should be used when a drug is unable to hit its pharmacological target. Thus, laboratory methods used to evaluate the effects of antiplatelet drugs should be designed to measure the direct pharmacodynamic effect of a drug, rather than the consequences for global platelet function. Based on physiological/pathophysiological, pharmacological, and practical considerations, the authors propose the following assays to be used to measure the effects of oral antiplatelet drugs: for the detection of aspirin actions, thromboxane or arachidonic acid-induced responses (light aggregometry, whole-blood aggregometry) should be measured; for the detection of clopidogrel actions, VASP (vasodilator-stimulated phosphoprotein) phosphorylation (flow cytometry) or ADP-(adenosine diphosphate-)induced responses (light aggregometry, whole-blood aggregometry, possibly also flow cytometry) should be measured.ZusammenfassungEs gibt zwei prinzipielle Gründe für die Messung der pharmakologischen Wirksamkeit von Thrombozytenfunktionshemmern: Auf der einen Seite kann eine unzureichende Hemmung der Thrombozytenfunktion zu atherothrombotischen Komplikationen führen; auf der anderen Seite kann eine exzessive Hemmung der Thrombozytenfunktion in Blutungskomplikationen resultieren. Die klinische Bedeutung der Messung der Wirkungen von Thrombozytenfunktionshemmern zeigt sich in einer zunehmenden Evidenz für die Assoziation einer fehlenden pharmakologischen Wirksamkeit mit thromboembolischen Ereignissen. Es wird häufig postuliert, dass es keine allgemein akzeptierte Definition für „Resistenz““ gegenüber Thrombozytenfunktionshemmern gibt. Stattdessen wird eine semantische Diskussion über den korrekten Begriff zur Beschreibung des Phänomens geführt. Aus pharmakologischer Sicht gibt es jedoch eine klare Definition der Resistenz gegenüber Thrombozytenfunktionshemmern: Der Begriff „Resistenz“ sollte benutzt werden, wenn ein Pharmakon den für die klinische Wirksamkeit entscheidenden pharmakodynamischen Mechanismus nicht ausüben kann. Daher sollten bevorzugt solche Labormethoden eingesetzt werden, die möglichst direkt den pharmakodynamischen Effekt eines Pharmakons erfassen. Basierend auf physiologischen/pathophysiologischen, pharmakologischen und praktischen Überlegungen wird vorgeschlagen, dass zur Erfassung der Wirkungen von Acetylsalicylsäure die Thromboxanbildung oder die arachidonsäureinduzierte Thrombozytenaggregation (Lichtaggregometrie, Vollblutaggregometrie) bestimmt werden sollte. Zur Erfassung der Wirkungen von Clopidogrel erscheint die Bestimmung der VASP-(„vasodilator-stimulated phosphoprotein“-)Phosphorylierung (Durchflusszytometrie) oder die ADP-(Adenosindiphosphat-) induzierte Thrombozytenaggregation (Lichtaggregometrie, Vollblutaggregometrie) bzw. Thrombozytenaktivierung (Durchflusszytometrie) sinnvoll.

Membrane environment rather than tissue factor expression determines thrombin formation triggered by monocytic cells undergoing apoptosis

Monocyte apoptosis is an important determinant of atherothrombosis. Two major mechanisms for apoptosis‐associated thrombogenicity have been described: exposure of negatively charged membrane phospholipids and up‐regulation of tissue factor (TF). However, the relative importance of these mechanisms is unclear. Thus, procoagulant functions (thrombin generation) of apoptotic (staurosporine, 2 μM, 24 h) U937 cells versus cell‐derived microparticles (MPs) were studied. In apoptotic U937 cells, a significant increase in TF mRNA (real‐time PCR), surface expression of TF (flow cytometry), and total cellular amount of TF (Western blotting) was observed. Control cells only minimally triggered thrombin generation (endogenous thrombin potential), and apoptotic cells were highly procoagulant. However, addition of negatively charged membranes completely restored the thrombin generation capacity of control U937 cells to the levels of apoptotic cells. MPs (defined as CD45+ particles of subcellular size), derived from apoptotic U937 cells, were highly procoagulant but did not exhibit an increased TF expression or annexin V binding. Taken together, our data support the concept that the membrane environment, independent of TF expression, determines the extent of thrombin formation triggered by apoptosis of monocytic cells. Externalization of negatively charged phospholipids represents the most important mechanisms for whole cells. Additional yet unknown mechanisms appear to be involved in the procoagulant actions of MPs derived from apoptotic monocytes.