Association of the phosphodiesterase 4D (PDE4D) gene and cardioembolic stroke in an Australian cohort

Background: Large-scale epidemiological studies support an important role for susceptibility genes in the pathogenesis of ischemic stroke, with phosphodiesterase 4D identified as the first gene predisposing to ischemic stroke. Several single nucleotide polymorphisms within the phosphodiesterase 4D gene have been implicated in the pathogenesis of stroke. Aim: Undertake a multivariate analysis of six single nucleotide polymorphisms within the phosphodiesterase 4D gene in a previously defined Australian stroke cohort, to determine whether these single nucleotide polymorphisms have an association with ischemic stroke. Methods: This case–control study was performed using an existing genetic database of 180 ischemic stroke patients and 301 community controls, evaluated previously for cerebrovascular risk factors (hypertension, hypercholesterolemia, diabetes, paroxysmal atrial fibrillation, smoking and history of stroke in a first-degree relative). Based on previously reported associations with large vessel disease, ischemic stroke, cardioembolic stroke or a mixture of these, six single nucleotide polymorphisms in the phosphodiesterase 4D gene were selected for study, these being single nucleotide polymorphisms 13, 19, rs152312, 45, 83 and 87, based on previously utilized DeCODE nomenclature. Single nucleotide polymorphisms were genotyped using a sequence-specific polymerase chain reaction method and gel electrophoresis. Logistic regression was undertaken to determine the relevance of each polymorphism to stroke. Further analysis was undertaken to determine the risk of stroke following stratification for stroke sub-type and etiology. Results: Significant odds ratios were found to be associated with cardioembolic strokes in two single nucleotide polymorphisms: rs152312 and SNP 45 (P<0·05). Conclusions: Our findings demonstrated an association between cardioembolic stroke and phosphodiesterase 4D single nucleotide polymorphisms rs152312 and 45. No significant association was found for the other four single nucleotide polymorphisms investigated within the phosphodiesterase 4D gene. We propose that the results from this Australian population support the concept that a large prospective international study is required to investigate the role of phosphodiesterase 4D in the cardiogenic cause of ischemic stroke.Austin G. Milton, Verna M. Aykanat, M. Anne Hamilton-Bruce, Mark Nezic, Jim Jannes, Simon A. Kobla

PDE8 Regulates Rapid Teff Cell Adhesion and Proliferation Independent of ICER

BACKGROUND: Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions. METHODOLOGY/PRINCIPAL FINDINGS: We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem-/- mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection. CONCLUSION/SIGNIFICANCE: Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells

Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling

Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1–10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium