217 MECHANISMS OF ACTION OF ESE1, A NOVEL TRANSCRIPTIONAL REGULATOR OF CARTILAGE REMODELING, IN MMP-13 REGULATION

Ischemic preconditioning (IPC) limits myocardial infarct size through the activation of the PI3K-Akt signal cascade; however, little is known about the roles of individual PI3K isoforms in cardioprotection. We aimed, therefore, to elucidate the role of the PI3K alpha isoform in cardioprotection Pharmacological PI3K alpha inhibition was assessed in isolated-perfused mouse hearts subjected to ischemia/reperfusion injury (IRI), either during the IPC procedure or at reperfusion. PI3K alpha inhibition abrogated the IPC-induced protective effect at reperfusion, but not when given only during the IPC protocol. These results were confirmed in an in vivo model. Moreover, pharmacological PI3K alpha activation by insulin at reperfusion was sufficient to confer cardioprotection against IRI. In addition, PI3K alpha was shown to be expressed and activated in mouse cardiomyocytes, mouse cardiac endothelial cells, as well as in mouse and human heart tissue. Furthermore, PI3K alpha was shown to mediate its effect though the inhibition of mitochondrial permeability transition pore opening. In conclusion, PI3K alpha activity is required during the early reperfusion phase to reduce myocardial infarct size. This suggests that strategies specifically enhancing the alpha isoform of PI3K at reperfusion promote tissue salvage and as such, and could provide a direct target for clinical treatment of IRI.Fundacion Rafael del Pino FONDECYT 3160298 British Heart Foundation Cancer Research UK C23338/A15965 UK NIHR University College London Hospitals Biomedical Research Centr

<em>In vivo</em> label-free structural and biochemical imaging of coronary arteries using an integrated ultrasound and multispectral fluorescence lifetime catheter system.

Existing clinical intravascular imaging modalities are not capable of accurate detection of critical plaque pathophysiology in the coronary arteries. This study reports the first intravascular catheter combining intravascular ultrasound (IVUS) with multispectral fluorescence lifetime imaging (FLIm) that enables label-free simultaneous assessment of morpho logical and biochemical features of coronary vessels in vivo. A 3.7 Fr catheter with a fiber-optic channel was constructed based on a 40 MHz clinical IVUS catheter. The ability to safely acquire co-registered FLIm-IVUS data in vivo using Dextran40 solution flushing was demonstrated in swine coronary arteries. FLIm parameters from the arterial wall were consistent with the emission of fluorophores present in healthy arterial wall (collagen, elastin). Additionally, structural and biochemical features from atherosclerotic lesions were acquired in ex vivo human coronary samples and corroborated with histological findings. Current results show that FLIm parameters linked to the amount of structural proteins (e.g. collagen, elastin) and lipids (e.g. foam cells, extracellular lipids) in the first 200 &mu;m of the intima provide important biochemical information that can supplement IVUS data for a comprehensive assessment of plaques pathophysiology. The unique FLIm-IVUS system evaluated here has the potential to provide a comprehensive insight into atherosclerotic lesion formation, diagnostics and response to therapy

Polyamine depletion inhibits NF-kappaB binding to DNA and interleukin-8 production in human chondrocytes stimulated by tumor necrosis factor-alpha.

The activation of the NF-kappaB pathway by pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFalpha), can be an important contributor for the re-programming of chondrocyte gene expression, thereby making it a therapeutic target in articular diseases. To search for new approaches to limit cartilage damage, we investigated the requirement of polyamines for NF-kappaB activation by TNFalpha in human C-28/I2 chondrocytes, using alpha-difluoromethylornithine (DFMO), a specific polyamine biosynthesis inhibitor. The NF-kappaB pathway was dissected by using pharmacological inhibitors or by expressing a transdominant IkappaBalpha super repressor. Treatment of C-28/I2 chondrocytes with TNFalpha resulted in a rapid enhancement of nuclear localization and DNA binding activity of the p65 NF-kappaB subunit. TNFalpha also increased the level and extracellular release of interleukin-8 (IL-8), a CXC chemokine that can have a role in arthritis, in an NF-kappaB-dependent manner. Pre-treatment of chondrocytes with DFMO, while causing polyamine depletion, significantly reduced NF-kappaB DNA binding activity. Moreover, DFMO also decreased IL-8 production without affecting cellular viability. Restoration of polyamine levels by the co-addition of putrescine circumvented the inhibitory effects of DFMO. Our results show that the intracellular depletion of polyamines inhibits the response of chondrocytes to TNFalpha by interfering with the DNA binding activity of NF-kappaB. This suggests that a pharmacological and/or genetic approach to deplete the polyamine pool in chondrocytes may represent a useful way to reduce NF-kappaB activation by inflammatory cytokines in arthritis without provoking chondrocyte apoptosis