Publications

<h3>Introduction:</h3> COPD is associated with cellular senescence, hallmarks of which include increased cell size, autofluorescence, senescence associated-β-galactosidase (SA-β-Gal) staining, mitochondrial dysfunction, reduced proliferation and cell cycle inhibitor expression. Dilution of these markers due to proliferation of non-senescent cells makes studying senescence in proliferative cells, such as fibroblasts, challenging. <h3>Aim:</h3> Assess whether senescent fibroblasts can be isolated from a mixed COPD fibroblast population. <h3>Method:</h3> Fibroblasts were isolated from COPD patient lung resections (n=6). Cells were sorted using a FACSAria Fusion into a "senescence-enriched population" (largest and most autofluorescence) and a "non-senescent population" (smallest, least autofluorescence). Senescence was investigated in these populations using SA-β-Gal for senescent cell identification, iCELLigence technology to assess proliferation, Seahorse assay to investigate mitochondrial function and RT-qPCR to measure cell cycle inhibitor expression. <h3>Results:</h3> Senescence-enriched fibroblasts were larger and more autofluorescence and displayed greater SA-β-Gal staining. These cells trended towards reduced proliferation over the first 48h of growth compared to the non-senescent population. Senescence-enriched fibroblasts showed increased basal mitochondrial function (p&lt;0.05), maximal respiration (p&lt;0.05) and ATP production (p=0.06). Senescence-enriched fibroblasts also exhibited elevated p16^INK4a (p&lt;0.05), p21^WAF1/CIP1 (p&lt;0.05) and p15^INK4b (p=0.10) gene expression. <h3>Conclusion:</h3> These data suggest FACS isolate senescent-enriched fibroblasts. This technique can be used to study fibroblast senescence mechanisms and provide therapeutic targets for COPD.

Abstract Abnormal expression of TGF-β1 is believed to play an important role in the pathogenesis of a number of chronic inflammatory and immune lung diseases, including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. Gene activation in eukaryotes requires coordinated use of specific cell signals, chromatin modifications, and chromatin remodeling. We studied the roles of the ubiquitous inflammatory transcription factors, NF-κB and AP-1, in activation of the TGF-β1 gene and histone acetylation at the TGF-β1 promoter. IL-1β-induced TGF-β1 protein secretion and mRNA expression were prevented by actinomycin D and were attenuated by the inhibitor of κB kinase 2 inhibitor AS602868 and the JNK inhibitor SP600125, suggesting a degree of transcriptional regulation mediated by the NF-κB and AP-1 pathways. We demonstrated that IL-1β activated the p65 subunit of NF-κB and the c-Jun subunit of AP-1. Using chromatin immunoprecipitation assays, we observed a sequential recruitment of p65 and c-Jun, accompanying ordered elevation of the levels of histone H4 and H3 acetylation and recruitment of RNA polymerase II at distinct regions in the native TGF-β1 promoter. The specific NF-κB and AP-1 binding sites in the TGF-β1 promoter were confirmed by an ELISA-based binding assay, and evidence for histone hyperacetylation in TGF-β1 induction was supported by the observation that the histone deacetylase inhibitor trichostatin A enhanced basal and IL-1β-induced TGF-β1 mRNA expression. Our results suggest that IL-1β-stimulated transcription of TGF-β1 is temporally regulated by NF-κB and AP-1 and involves histone hyperacetylation at distinct promoter sites.

Vulnerable plaquesThe majority of coronary thrombi ( 75%) is caused by plaque rupture. 1,2Prototype of the rupture-prone plaque contains a large, soft, lipid-rich necrotic core with a thin and inflamed fibrous cap, so-called thin-cap fibroatheroma (TCFA) (Figure 1). 3,4Other common features include expansive remodelling, large plaque size, plaque haemorrhage, neovascularization, adventitial inflammation, and 'spotty' calcifications. 4Thin-cap fibroatheroma caps are usually ,65 mm thick. 4Figure 2 summarizes factors contributing to the formation of vulnerable plaques.No distinct morphological features have been identified for the erosion-prone plaques, but they are usually rarely associated with expansive remodelling, scarcely calcified, and contain only limited inflammation. 2,5 Inflammatory cells, cytokines, chemokines, and growth factorsVulnerable plaques contain monocytes, macrophages, and T-cells.Of the T-cells, CD4+ T-helper (Th) cells are the most prominent. 6-cells can differentiate into a Th1 phenotype, which secretes and responds to IFN-g or a Th2 phenotype, which secretes and responds to IL-4, IL-10, and IL-13 (Figure 3).T-cells promote the vulnerability of plaques through their effects on macrophages.Similarly, there are two main plaque macrophage phenotypes: pro-inflammatory M1 macrophages (IFN-g-induced) and anti-inflammatory or regulatory M2 macrophages (IL-4/IL-13-induced). 7 Cytokines and chemokines important for regulating inflammatory and immune responses are listed in Supplementary material online,

The additional information provided by 3-D reconstruction of intracoronary ultrasound images (ICUS) during stent implantation, was assessed in 8 pts. In total 14 stents were used: 6 Palmaz-Schatz, 3 AVE and 5 Wallstent. The images of all 14 coronary segments encompassing the full stent length and the adjacent reference segments were acquired by using a motorized pullback at a constant speed (1mm/s) and immediately processed in the interventional suite using an automated contour detection algorithm based on acoustic quantification (Figure). Residual percent area stenosis (%AS) of the stented segment, automatically measured on-line after 3-D reconstruction, was compared with ICUS measurements obtained by direct review of the video-tape. In three pts studied before stent implantation on-line 3-D reconstruction facilitated the selection of the stent length by measuring length of dissection and distance between stenosis and left main or diagonal branch. After stent implantation, the absence of stent overlapping in three patients receiving multiple stents and the relationship with side branches could be assessed. The visual information from the 3-D longitudinal views and the on-line measurements triggered additional balloon inflations in 5pts. The selection of frames and the measurement from tape required a longer time than 3-D reconstruction and measurement (4.4 vs 2.35 min. p &lt; 0.0011 and resulted in a slight underestimation of the residual %AS with 3 pts showing a &gt;20% %AS with 3-D not recognized from tape analysis. Conclusion Online 3-D reconstruction of ICUS for intracoronary stenting facilitates stent selection, evaluates adequate stent deployment by rapidly and accurately measuring residual intrastent stenosis and detecting its relationship with anatomic landmarks.

The clinical efficacy and safety of directional coronary atherectomy for the treatment of stable and unstable angina were assessed in 82 patients with stable and 68 patients with unstable angina. Therefore, clinical and angiographic follow-up was obtained in a prospectively collected consecutive series of 150 atherectomy procedures. Restenosis was assessed clinically and by quantitative angiography. The overall clinical success rate of atherectomy for patients with unstable and stable angina was 88% and 91%, respectively. No significant differences were found for in-hospital event rates between the unstable and stable angina groups: death (1.5% vs 0%), myocardial infarction (10% vs 6%), and emergency bypass operation (3% vs 2%). These clinical events were related to the occurrence of abrupt occlusions (8.8% in patients with stable and 6.1% in those with unstable angina; p = NS). Clinical follow-up was achieved in 100% of the patients with stable and unstable angina at a mean interval of 923 and 903 days, respectively. Two-year survival rates were 96% and 97% in the populations with unstable and stable angina, respectively. There were no significant differences with respect to bypass surgery and angioplasty, but event-free survival at 2 years was significantly lower in the unstable (54%) than the stable (69%) angina group. Quantitative coronary angiography did not detect any difference in luminal renarrowing during the 6-month angiographie follow-up period. Although directional coronary atherectomy can be performed effectively in patients with unstable and stable angina, the long-term clinical outcome was less favorable in the unstable angina group. The overall higher incidence of adverse events in patients with unstable angina occurred despite the excision of unstable plaque material, and may therefore reflect the inherent instability of the syndrome rather than the inability of atherectomy to establish a persistent success.

No abstract is provided for this article.

Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline.To determine the role of phosphoinositide-3-kinase-delta (PI3K-δ) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline.Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-α-induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n = 17) and compared with that of nonsmoking (n = 8) and smoking (n = 7) control subjects. The effect of theophylline and a selective PI3K-δ inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke-exposed mice.Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 ± 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 ± 10.5 nM; P = 0.018) and smoking control subjects (47.5 ± 19.6 nM; P = 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-δ, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-δ failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette-smoke-exposed mice in vivo.Inhibition of oxidative stress dependent PI3K-δ activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.