To better understand the molecular basis of chronic obstructive pulmonary disease (COPD), we used serial analysis of gene expression (SAGE) and microarray analysis to compare the gene expression patterns of lung tissues from COPD and control smokers. A total of 59,343 tags corresponding to 26,502 transcripts were sequenced in SAGE analyses. A total of 327 genes were differentially expressed (1.5-fold up-or down-regulated). Microarray analysis using the same RNA source detected 261 transcripts that were differentially expressed to a significant degree between GOLD-2 and GOLD-0 smokers. We confirmed the altered expression of a select number of genes by using real-time quantitative RT-PCR. These genes encode for transcription factors (EGR1 and FOS), growth factors or related proteins (CTGF, CYR61, CX3CL1, TGFB1, and PDGFRA), and extracellular matrix protein (COL1A1). Immunofluorescence studies on the same lung specimens localized the expression of Egr-1, CTGF, and Cyr61 to alveolar epithelial cells, airway epithelial cells, and stromal and inflammatory cells of GOLD-2 smokers. Cigarette smoke extract induced Egr-1 protein expression and increased Egr-1 DNA-binding activity in human lung fibroblast cells. Cytomix (tumor necrosis factor ␣, IL-1␤, and IFN-␥) treatment showed that the activity of matrix metalloproteinase-2 (MMP-2) was increased in lung fibroblasts from EGR1 control (؉/؉) mice but not detected in that of EGR1 null (؊/؊) mice, whereas MMP-9 was regulated by EGR1 in a reverse manner. Our study represents the first comprehensive analysis of gene expression on GOLD-2 versus GOLD-0 smokers and reveals previously unreported candidate genes that may serve as potential molecular targets in COPD.

Subtle clotting that occurs on the luminal surface of atherosclerotic plaques presents a novel target for nanoparticle-based diagnostics and therapeutics. We have developed modular multifunctional micelles that contain a targeting element, a fluorophore, and, when desired, a drug component in the same particle. Targeting atherosclerotic plaques in ApoE-null mice fed a high-fat diet was accomplished with the pentapeptide cysteine-arginine-glutamic acid-lysine-alanine, which binds to clotted plasma proteins. The fluorescent micelles bind to the entire surface of the plaque, and notably, concentrate at the shoulders of the plaque, a location that is prone to rupture. We also show that the targeted micelles deliver an increased concentration of the anticoagulant drug hirulog to the plaque compared with untargeted micelles.cysteine-arginine-glutamic acid-lysine-alanine ͉ hirulog ͉ plaque ͉ imaging ͉ nanoparicles C ardiovascular disease affects 1 in 3 people in the United States during their lifetime, and accounts for nearly a third of the deaths that occur each year (1). Atherosclerosis is one of the leading causes of cardiovascular disease, and it results in raised plaques in the arterial wall that can occlude the vascular lumen and block blood flow through the vessel. Recently, it has become clear that not all plaques are the same. Those susceptible to rupture, fissuring, and subsequent thrombosis are most frequently the cause of acute coronary syndromes and death (2).Rupture of an atherosclerotic plaque exposes collagen and other plaque components to the bloodstream. This rupture initiates hemostasis in the blood vessel and leads to activation of thrombin and a thrombus to form at the site of rupture. Elevated levels of activated thrombin bound to the vessel wall have been observed up to 72 h after vascular injury (3). These elevated thrombin levels not only induce clot formation but also have been implicated in the progression of atherosclerosis by causing smooth muscle cells to bind circulating low density lipoprotein (4). Subtle clotting in plaques is also indicated by deposition of fibrin(ogen) both inside and on the surface of atherosclerotic plaques, which has been well documented since the 1940s (5-7).Fibrin-containing blood clots have been extensively used as a target for site-specific delivery of imaging agents and anticlotting agents to thrombi (8-10). Delivering anticoagulants into vessels where clotting is taking place has been shown to be effective at reducing the formation and expansion of clots, and it also decreases the risk of systemic side effects (11,12). Antibodies and peptides that bind to molecular markers specifically expressed on atherosclerotic plaques have shown promise for plaque imaging in vivo (13-16), but clotting on the plaque has not been used as a target. We reasoned that the fibrin deposited on plaques could serve as a target for delivering diagnostic and therapeutic compounds to plaques.We chose the clot-binding peptide cysteine-arginine-glutamic acid-lysine-alanine (CREKA) to te...

Thirty eccentric contractions (ECs) were imposed upon rat dorsiflexors (n= 46) by activating the peroneal nerve and plantarflexing the foot ≈40 deg, corresponding to a sarcomere length change over the range 2.27‐2.39 μm for the tibialis anterior and 2.52‐2.66 μm for the extensor digitorum longus. Animals were allowed to recover for one of 10 time periods ranging from 0.5 to 240 h, at which time muscle contractile properties, immunohistochemical labelling and gene expression were measured. Peak isometric torque dropped significantly by ≈40 % from an initial level of 0.0530 ± 0.0009 Nm to 0.0298 ± 0.0008 Nm (P < 0.0001) immediately after EC, and then recovered in a linear fashion to control levels 168 h later. Immunohistochemical labelling of cellular proteins revealed a generally asynchronous sequence of events at the cellular level, with the earliest event measured being loss of immunostaining for the intermediate filament protein, desmin. Soon after the first signs of desmin loss, infiltration of inflammatory cells occurred, followed by a transient increase in membrane permeability, manifested as inclusion of plasma fibronectin. The quantitative polymerase chain reaction (QPCR) was used to measure transcript levels of desmin, vimentin, embryonic myosin heavy chain (MHC), myostatin, myoD and myogenin. Compared to control levels, myostatin transcripts were significantly elevated after only 0.5 h, myogenic regulatory factors significantly elevated after 3 h and desmin transcripts were significantly increased 12 h after EC. None of the measured parameters provide a mechanistic explanation for muscle force loss after EC. Future studies are required to investigate whether there is a causal relationship among desmin loss, increased cellular permeability, upregulation of the myoD and desmin genes, and, ultimately, an increase in the desmin content per sarcomere of the muscle.

Screening of a phage library for peptides that bind to clotted plasma in the presence of liquid plasma yielded two cyclic decapeptides, CGLIIQKNEC (CLT1) and CNAGESSKNC (CLT2). When injected intravenously into mice bearing various types of tumors, fluorescein-conjugated CLT peptides accumulated in a fibrillar meshwork in the extracellular compartment of the tumors, but were not detectable in other tissues of the tumor-bearing mice. The tumor homing of both peptides was strongly reduced after coinjection with unlabeled CLT2, indicating that the two peptides recognize the same binding site. The CLT peptide fluorescence colocalized with staining for fibrin(ogen) present in the extravascular compartment of tumors, but not in other tissues. The CLT peptides did not home to tumors grown in fibrinogen-null mice or in mice that lack plasma fibronectin. The CLT peptides also accumulated at the sites of injury in arteries, skeletal muscle, and skin. We conclude that the CLT peptides recognize fibrin-fibronectin complexes formed by clotting of plasma proteins that have leaked into the extravascular space in tumors and other lesions. These peptides may be useful in targeting diagnostic and therapeutic materials into tumors and injured tissues.fibronectin ͉ imaging ͉ phage display ͉ tumor targeting ͉ fibrin

BCR/ABL, the oncoprotein responsible for chronic myeloid leukemia (CML), transforms hematopoietic cells through both Ras-dependent and -independent mechanisms. Farnesyl protein transferase inhibitors (FTIs) were designed to block mutant Ras signaling, but they also inhibit the growth of transformed cells with wildtype Ras, implying that other farnesylated targets contribute to FTI action. In the current study, the clinical candidate FTI SCH66336 was characterized for its ability to inhibit BCR/ABL transformation. When tested against BCR/ABL-BaF3 cells, a murine cell line that is leukemogenic in mice, SCH66336 potently inhibited soft agar colony formation, slowed proliferation, and sensitized cells to apoptotic

We followed 123 patients with primary intracerebral hemorrhage (ICH), defined as bleeding without known precipitating cause except hypertension, for an average of 4.6 years or until death in order to determine the incidence, prevalence, and type of epileptic seizures. Twenty-five percent had seizures. In one-half of these, the seizures began within 24 hours of the hemorrhage. Survival table analysis predicted a potential cumulative seizure incidence of 50%, had all patients survived 5 years. Seizure incidence was high with bleeding into lobar cortical structures (54%), low with basal ganglionic hemorrhages (19%), and zero with thalamic hemorrhages. Within the basal ganglia, caudate involvement predicted seizures; within the cortex, temporal or parietal involvement predicted seizures. Although seizure incidence was high, prevalence of chronic epilepsy was much lower: 13% in 30-day to 2-year survivors and 6.5% in 2- to 5-year survivors. Seizure incidence is higher than previously reported after ICH because small lobar hemorrhages are the most epileptogenic and are now easily recognized with computed tomography.

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The purpose of this study was to determine the influence of age, sex, and strength training (ST) on large-scale gene expression patterns in vastus lateralis muscle biopsies using high-density cDNA microarrays and quantitative PCR. Muscle samples from sedentary young (20-30 yr) and older (65-75 yr) men and women (5 per group) were obtained before and after a 9-wk unilateral heavy resistance ST program. RNA was hybridized to cDNA filter microarrays representing ~4,000 known human genes and comparisons were made among arrays to determine differential gene expression as a result of age and sex differences, and/or response to ST. Sex had the strongest influence on muscle gene expression, with differential expression (>1.7-fold) observed for ~200 genes between men and women (~75% with higher expression in men). Age contributed to differential expression as well, as ~50 genes were identified as differentially expressed (>1.7-fold) in relation to age, representing structural, metabolic, and regulatory gene classes. Sixty-nine genes were identified as being differentially expressed (>1.7-fold) in all groups in response to ST, and the majority of these were downregulated. Quantitative PCR was employed to validate expression levels for caldesmon, SWI/ SNF (BAF60b), and four-and-a-half LIM domains 1. These significant differences suggest that in the analysis of skeletal muscle gene expression issues of sex, age, and habitual physical activity must be addressed, with sex being the most critical variable. Keywordsaging; exercise; gender; transcription; transcriptome THE LOSS OF SKELETAL MUSCLE mass and strength with advancing age is associated with frailty, loss of function, and the deterioration of health status in the elderly (13,33), and these changes may be influenced by sex differences (18,20,28,35). The potential of strength training (ST) to reverse age-associated losses of muscle mass and strength in both men and women is well established (9,17,34); however, little is known about why muscle mass is lost with age or how muscle responds to ST, at the molecular level.Recent evidence has indicated that changes in gene expression with advancing age may contribute to a deterioration in muscle function (11,16,19,21,25). Similarly, limited evidence Address for reprint requests and other correspondence: S. M. Roth, A300 Crabtree Hall-GSPH, Dept. Human Genetics, Univ. of Pittsburgh, Pittsburgh, PA 15261 (sroth@hgen.pitt.edu).. NIH Public Access Author ManuscriptPhysiol Genomics. Author manuscript; available in PMC 2010 January 27. Published in final edited form as:Physiol Genomics. ; 10(3): 181-190. doi:10.1152/physiolgenomics.00028.2002. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript supports the importance of differences in gene expression in explaining muscle phenotype differences between men and women (7,31,37). Changes in gene expression have been wellcharacterized for a number of muscle-specific genes in response to acute resistance-type exercise (3). A general limitation to previous ...