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Chronic degenerative inflammatory diseases, such as chronic obstructive pulmonary disease and Alzheimer's 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.

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.

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.

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 19 F magnetic resonance imaging, together with α2-antiplasmin peptide (α2 AP )–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 1 H and 19 F 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, α2 AP -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 α2 AP -PFCs >60 minutes after thrombus induction no longer resulted in detectable 19 F magnetic resonance imaging signals. Corresponding results were obtained in ex vivo generated human clots. Thus, α2 AP -PFCs can visualize freshly developed thrombi that might still be susceptible to pharmacological intervention. Conclusions— Our results demonstrate that 1 H/ 19 F magnetic resonance imaging, together with α2 AP -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 19 F magnetic resonance imaging.

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) via endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of red cell eNOS compared to EC eNOS for vascular hemodynamics and NO metabolism. Methods: We generated tissue-specific "loss-" and "gain-of-function" models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created two founder lines carrying a floxed eNOS (eNOS flox/flox ) for Cre-inducible knock out (KO), as well as gene construct with an inactivated floxed/inverted exon (eNOS inv/inv ) for a Cre-inducible knock in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or endothelial cells (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and NO metabolism were compared ex vivo and in vivo . Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation (FMD), and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or FMD but were hypertensive. Treatment with the NOS inhibitor L-NAME further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. While both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs as compared to EC eNOS KOs. Crucially, reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOS inv/inv mice, while the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.

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 ␣ v ␤ 3 -agonist, RGDS, latrunculin A, and an inhibitor of Rho-activated kinase inhibited HAS2 expression. Conversely, blocking antibodies to ␣ v ␤ 3 abolished the down-regulation of HAS2 and the cytoskeletal effects. Furthermore, inhibition of cofilin phosphorylation in response to CF was prevented by ␣ v ␤ 3 -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 ␣ v ␤ 3 -blocking antibodies. In conclusion, these data suggest that CF activate ␣ v ␤ 3 -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.

BACKGROUND: It was recently found that cAMP mediates protein kinase A-independent effects through Epac proteins. The aim of this study was to investigate the role of Epac in migration and proliferation of prostate carcinoma cells. METHODS: The effect of Epac activation was determined by [ 3 H]thymidine incorporation and scratch assays in PC-3 and DU 145 cells. Furthermore, cytoskeletal integrity was analysed by phalloidin staining. The participation of intracellular Epac effectors such as mitogen-activated protein (MAP) kinases, Rap1-and Rho-GTPases was determined by immunoblotting and pull-down assay. RESULTS: The specific Epac activator 8-pCPT-2 0 -O-Me-cAMP (8-pCPT) interfered with cytoskeletal integrity, reduced DNA synthesis, and migration. Although 8-pCPT activated Rap1, it inhibited MAP kinase signalling and RhoA activation. These findings were translated into functional effects such as inhibition of mitogenesis, cytoskeletal integrity, and migration. CONCLUSION: In human prostate carcinoma cells, Epac inhibits proliferative and migratory responses likely because of inhibition of MAP kinase and RhoA signalling pathways. Therefore, Epac might represent an attractive therapeutic target in the treatment of prostate cancer.