Immunohistochemistry of 8-OHdG nuclear labeling of the RPE.

<p>8 month C57Bl6 mouse exposed to smoke for 6 months showing A. DAPI labeled nuclei (arrows); B. 8-OHdG labeled RPE nuclei (arrows); C. Merged image of A and B with the Brightfield image showing violet nuclei (arrows); D. Merged image of DAPI and IgG1 control image with Brightfield image overlay. 8 month old C57Bl6 mouse raised in air showing DAPI labeled RPE nuclei in E and 8-OHdG immunostaining in F; G. Merged DAPI and 8-OHdG immunostained image with Brightfield image overlay showing blue nuclei in H. Brightfield image. RPE, retinal pigmented epithelium; Ch, choroid. Bar = 15 μm. Figure shows representative images from N = 10 mice (50 samples/mouse).</p

Patient characteristics.

<p>FEV₁ = Forced expiratory volume at 1 sec; FVC = Function vital capacity; SD = Standard deviation; COPD = Chronic obstructive pulmonary disease.</p><p>GOLD (Global Initiative for Chronic Obstructive Lung Disease) Stages:</p><p>1 – mild COPD: FEV₁≥80% predicted, FEV₁:FVC<70%.</p><p>2 – moderate COPD: 50%≤FEV₁≤80% predicted, FEV₁:FVC<70%.</p><p>3 – severe COPD: 30%≤FEV₁≤50% predicted, FEV₁:FVC<70%.</p><p>4 – very severe COPD: FEV1<30%predicted or FEV₁<50% predicted with chronic respiratory failure, FEV₁:FVC<70%.</p><p>Pack years: (Packs smoked per day)×(years as a smoker).</p

Transmission electron microscopy of Bruch membrane of mice exposed to air (A) or cigarette smoke (B,C).

<p>A. Mouse exposed to air shows regular basal infoldings (BI) of the RPE and unthickened Bruch membrane (BrM). B. The RPE show loss of basal infoldings. Bruch membrane is thickened due to an outer collagenous layer deposit (OCL). C. The RPE show more severe loss of basal infoldings than in (B). Bruch membrane contains early basal laminar deposits (*), OCL, and choriocapillaris (CC) basement membrane reduplication (arrows). Bar = 500 nm.</p

Suppressed Expression of T-Box Transcription Factors Is Involved in Senescence in Chronic Obstructive Pulmonary Disease

<div><p>Chronic obstructive pulmonary disease (COPD) is a major global health problem. The etiology of COPD has been associated with apoptosis, oxidative stress, and inflammation. However, understanding of the molecular interactions that modulate COPD pathogenesis remains only partly resolved. We conducted an exploratory study on COPD etiology to identify the key molecular participants. We used information-theoretic algorithms including Context Likelihood of Relatedness (CLR), Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Inferelator. We captured direct functional associations among genes, given a compendium of gene expression profiles of human lung epithelial cells. A set of genes differentially expressed in COPD, as reported in a previous study were superposed with the resulting transcriptional regulatory networks. After factoring in the properties of the networks, an established COPD susceptibility locus and domain-domain interactions involving protein products of genes in the generated networks, several molecular candidates were predicted to be involved in the etiology of COPD. These include COL4A3, CFLAR, GULP1, PDCD1, CASP10, PAX3, BOK, HSPD1, PITX2, and PML. Furthermore, T-box (TBX) genes and cyclin-dependent kinase inhibitor 2A (CDKN2A), which are in a direct transcriptional regulatory relationship, emerged as preeminent participants in the etiology of COPD by means of senescence. Contrary to observations in neoplasms, our study reveals that the expression of genes and proteins in the lung samples from patients with COPD indicate an increased tendency towards cellular senescence. The expression of the anti-senescence mediators TBX transcription factors, chromatin modifiers histone deacetylases, and sirtuins was suppressed; while the expression of TBX-regulated cellular senescence markers such as CDKN2A, CDKN1A, and CAV1 was elevated in the peripheral lung tissue samples from patients with COPD. The critical balance between senescence and anti-senescence factors is disrupted towards senescence in COPD lungs.</p> </div

TUNEL labeling of RPE cells from mice exposed to cigarette smoke for 6 months.

<p>A. TUNEL labeled (red) RPE nuclei are indicated by the arrows. B. Nuclei are stained with DAPI (blue), as labeled by the arrows. C. Merged image of A and B separating TUNEL from DAPI only stained nuclei. D. Brightfield image of the RPE, choroid (Ch) and sclera (S). E. Merged image of a mouse raised in air for 6 months. Arrows point to blue DAPI without red TUNEL labeling. Bar = 15 μm.</p

Several chromosome 2 genes linked to senescence hubs in transcriptional regulatory network fall within COPD susceptibility locus, 2q33.3–2q37.2.

<p>Genes in bold character are in or border the region of the susceptibility locus.</p

Several aging-related genes are statistically linked to the expressions of TBX2, CDKN2A, and TGFB1.

<p>Several aging-related genes are statistically linked to the expressions of TBX2, CDKN2A, and TGFB1.</p

Figure 4

<p>A) The state of the Type IV collagen alpha 3 subunit, COL4A3, depends on the states of both TBX2 and CDKN2A in human lung epithelial cells. Following Robust Multi-Array Analysis of a compendium of 109 Affymetrix arrays on the U133A platform, the Context Likelihood of Relatedness (CLR) algorithm was used to generate a transcriptional regulatory network involving all available probe sets (at a CLR likelihood estimate cut-off of 2.5). Olive-green nodes represent genes whose median probe set expressions are suppressed in COPD. White nodes represent genes whose median probe set expressions are elevated in COPD. COL4A3, whose expression is suppressed in the COPD lung, is thus statistically dependent on both TBX2 and CDKN2A. B) Evolutionarily conserved probable protein domain-domain interactions corresponding to the predictions of <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002597#pcbi-1002597-t005" target="_blank">Table 5</a>. The thickness of each edge is commensurate with the corresponding computed probabilities. The COL4A3 protein has the Collagen domain (Collagen in Pfam database; InterPro Database Accession IPR008160) and probably engages PML via its zf-C3HC4 domain (zf-C3HC4 in Pfam database; InterPro Database Accession IPR001841). By way of its Collagen domain, COL4A3 interacts with PITX2 via its Homeobox domain (Homeobox in Pfam database; InterPro Database Accession IPR001356). Among others, there is also a probable interaction between the zf-C3HC4 of PML and the Ankyrin repeat (Ank in Pfam database; InterPro Database Accession IPR002110) domain of CDKN2A that could impact the COPD etiology.</p

Inferelator predictions of regulators of twenty Biclusters generated using FABIA.

<p>Inferelator predictions of regulators of twenty Biclusters generated using FABIA.</p

Quantitative PCR data indicate TBX3, TBX5, HDAC6, SIRT1, SIRT6 gene expression is suppressed in the lungs of patients with COPD, while mRNA expression of HDAC2 does not change compared to lung tissue from normal smokers.

<p>The extent of the suppression is highly significant between patients with mild and those with severe COPD. Patient diagnosis was based on the National Heart, Lung, and Blood Institute/World Health Organization Global Initiative for Chronic Obstructive Lung Disease (GOLD) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002597#pcbi.1002597-Pauwels1" target="_blank">[146]</a>. Fifteen normal, nine mild COPD, and six severe COPD samples were used for this analysis. The data is represented as Mean ± S.D. The data was analyzed using student's t-test for comparing mRNA expression in the respective groups. *represents a significance of p-value<0.01.</p