In dystrophic muscle, an increase in reactive oxygen species (ROS) production and sarcolemmal calcium (Ca 2þ) influx contributes to stretch-induced muscle damage however mechanistic insights into the activation of these pathways is lacking. In mdx myofibers (murine Duchenne muscular dystrophy), we have demonstrated that with mechanical stretch, the microtubule (MT) cytoskeleton is a critical mechano-transduction element for the activation of NADPH oxi-dase2 (Nox2) derived ROS production; a pathway we term X-ROS signaling [1]. Downstream, we showed that X-ROS sensitized stretch activated channels (SACs) to increase sarcolemmal Ca 2þ influx during stretch. The significance of the MT cytoskeleton activation of X-ROS in mdx was revealed when the acute targeting of MT density proffered protection from contraction induced damage. In mammalian cells, the MT network is a dynamic structure in which MT density is determined by the stability of MT filaments. Our initial studies used acute pharmacological stabilization (taxol) or destabilization (colchicine) to establish MT network density as critical for the mechano-activation of X-ROS. We now interrogate critical upstream pathways and use new pharmacological and molecular approaches to explore the role of endogenous modulators of MT stability and how they may contribute to the enhanced X-ROS in dystrophic skeletal muscle.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O(2)(•-), H(2)O(2), and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O(2)(•-) and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O(2)(•-) that are important in the host defense mechanism. Although paracrine-derived O(2)(•-) plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca(2+) mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O(2)(•-) to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O(2)(•-) lead to calcium signaling in adjacent endothelial cells.

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Endoplasmic reticulum (ER) stress, a condition caused by accumulation of unfolded or misfolded proteins in the ER lumen, is Rationale: heightened in the COPD lung as a result of impaired protein degradation. Normally, a highly sophisticated, compensatory response termed the unfolded protein response (UPR) relieves ER stress by up-regulating expression of a multi-gene program which codes for molecules that regulate protein folding, transport, degradation and translation. The present study examined UPR gene expression in cultured human type I and II pneumocytes in which ER stress was induced by thapsigargin, a SERCA pump inhibitor which depletes ER calcium. We also examined UPR gene expression in the lungs of subjects with advanced COPD (GOLD 4, n=24), moderate COPD (GOLD 2, n=12), and subjects at risk but without COPD (GOLD 0, n=11).Pneumocytes were treated with 1μM thapsigargin for 24 hr. Lung tissues were obtained from the Temple University Tissue Bank Methods: and the NIH Lung Tissue Research Consortium. Total RNA from cells and tissues was isolated with TRI reagent (Sigma-Aldrich) and purified by RNeasy column (Qiagen). Within each GOLD group, individual RNA samples were assayed for integrity (Bioanalyzer 2100, Agilent) and pooled in equal amounts. UPR gene expression was assessed using a commercially available RT kit and 84 gene PCR array (RT² Profiler PCR Array system, SABiosciences).Thapsigargin treatment increased (> 2-fold up-regulation) expression of 15 UPR genes in cultured type I cells and 22 genes in Results: type II pneumocytes, including the canonical UPR effectors i.e., the chaperones, GRP78, calreticulin and calnexin; the foldase phosphodiesterase isomerase (PDI); and the apoptosis-inducing transcription factor, CHOP. In contrast to thapsigargin-treated pneumocytes, GRP78, calnexin, calreticulin, and PDI mRNA levels were similar in lung tissue obtained from all 3 GOLD groups. However, CHOP mRNA was increased 1.8 fold in the GOLD 2 group compared to GOLD 0 and GOLD 4. This increase was validated (p<0.02 by ANOVA) by RT-PCR of all individual samples.These data indicate that a canonical UPR can be induced in vitro in human pneumocytes. In contrast, a UPR does not seem to Conclusion: occur in the lungs of subjects with advanced COPD. Failure to express a classic UPR chaperone response may contribute to heightened ER stress, CHOP up-regulation and pneumocyte apoptosis in COPD.