p22phox C242T Single-Nucleotide Polymorphism Inhibits Inflammatory Oxidative Damage to Endothelial Cells and Vessels.

BACKGROUND: The NADPH oxidase, by generating reactive oxygen species, is involved in the pathophysiology of many cardiovascular diseases and represents a therapeutic target for the development of novel drugs. A single-nucleotide polymorphism, C242T of the p22(phox) subunit of NADPH oxidase, has been reported to be negatively associated with coronary heart disease and may predict disease prevalence. However, the underlying mechanisms remain unknown. METHODS AND RESULTS: With the use of computer molecular modeling, we discovered that C242T single-nucleotide polymorphism causes significant structural changes in the extracellular loop of p22(phox) and reduces its interaction stability with Nox2 subunit. Gene transfection of human pulmonary microvascular endothelial cells showed that C242T p22(phox) significantly reduced Nox2 expression but had no significant effect on basal endothelial O2 (.-) production or the expression of Nox1 and Nox4. When cells were stimulated with tumor necrosis factor-α (or high glucose), C242T p22(phox) significantly inhibited tumor necrosis factor-α-induced Nox2 maturation, O2 (.-) production, mitogen-activated protein kinases and nuclear factor κB activation, and inflammation (all P<0.05). These C242T effects were further confirmed using p22(phox) short-hairpin RNA-engineered HeLa cells and Nox2(-/-) coronary microvascular endothelial cells. Clinical significance was investigated by using saphenous vein segments from non-coronary heart disease subjects after phlebotomies. TT (C242T) allele was common (prevalence of ≈22%) and, in comparison with CC, veins bearing TT allele had significantly lower levels of Nox2 expression and O2 (.-) generation in response to high-glucose challenge. CONCLUSIONS: C242T single-nucleotide polymorphism causes p22(phox) structural changes that inhibit endothelial Nox2 activation and oxidative response to tumor necrosis factor-α or high-glucose stimulation. C242T single-nucleotide polymorphism may represent a natural protective mechanism against inflammatory cardiovascular diseases.

Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

RATIONALE: The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. OBJECTIVES: To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. METHODS: We used the gp130F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. MEASUREMENTS AND MAIN RESULTS: Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. CONCLUSIONS: Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

IL-6/Stat3-driven pulmonary inflammation, but not emphysema, is dependent on interleukin-17A in mice.

BACKGROUND AND OBJECTIVE: Pulmonary emphysema is linked to T cell-mediated autoimmune inflammation, although the pathogenic role of specific pro-inflammatory cytokines remains unclear. The Th17 type response, characterized by the production of the cytokine interleukin (IL)-17A, is modulated in part by the IL-6/signal transducer and activator of transcription (Stat)3 signalling axis and is associated with numerous autoimmune diseases. We therefore evaluated a causal role for IL-17A in the IL-6-driven gp130(F/F) mouse model for spontaneous pulmonary inflammation and emphysema. METHODS: The expression of Th17-related factors was quantified in the lungs of gp130(F/F) mice and emphysematous patients, and the degree of pulmonary inflammation and emphysema was measured in gp130(F/F) : Il17a-/- mice by immunohistochemistry, stereology and respiratory mechanics. RESULTS: In gp130(F/F) mice, lung gene expression of Il17a and other Th17-related factors was augmented compared with gp130+/+ (wild-type), gp130(F/F) : Il6-/- and gp130(F/F) : Stat3-/+ mice displaying normalized Stat3 activity and no lung inflammation. Importantly, genetic ablation of Il17a in gp130(F/F) : Il17a-/- mice prevented lung inflammation; however, emphysema still developed. Additionally, messenger RNA expression of inflammatory genes Cxcl1, Cxcl2, Ccl2 and Tnfα; as well as Il6 and the Stat3-target gene, Socs3, were upregulated in the lungs of gp130(F/F) mice compared with gp130(F/F) : Il17a-/- and gp130+/+ mice. Consistent with these findings, augmented IL17A expression was observed in emphysema patients presenting with inflammation compared with inflammation-free individuals. CONCLUSIONS: Collectively, our data suggest that the integration of IL-17A into the IL-6/Stat3 signalling axis mediates lung inflammation, but not emphysema, and that discrete targeting of IL-17A may alleviate pulmonary inflammatory-related diseases.

Cellular and molecular investigations into the development of the pectoral girdle.

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

Regulation of cell cycle and stress responses to hydrostatic pressure in fission yeast.

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress.

Inhibition of human mesangial cell proliferation by targeting T-type calcium channels.

BACKGROUND: Aberrant glomerular mesangial cell (MC) proliferation is a common finding in renal diseases. T-type calcium channels (T-CaCN) play an important role in the proliferation of a number of cell types, including vascular smooth muscle cells. The hypothesis that T-CaCN may play a role in the proliferation of human MC was investigated. METHODS: The presence of T-CaCN in primary cultures of human MC was examined using voltage clamping and by RT-PCR. The effect of calcium channel inhibitors, and of siRNA directed against the Cav3.2 T-CaCN isoform, on MC proliferation was assessed using the microculture tetrazolium assay and nuclear BrdU incorporation. RESULTS: Human MC express only the Cav3.2 T-CaCN isoform. Co-incubation of MC with a T-CaCN inhibitor (mibefradil, TH1177 or Ni(2+)) results in a concentration-dependent attenuation of proliferation. This effect cannot be attributed to direct drug-induced cytotoxicity or apoptosis and is not seen with verapamil, an L-type channel blocker. Transfection of MC with siRNA results in knockdown of T-CaCN Cav3.2 mRNA and a clear attenuation of MC proliferation. CONCLUSIONS: These results demonstrate for the first time an important role for T-CaCN in human MC proliferation. This could potentially lead to a novel therapy in the treatment of proliferative renal diseases.

Reprogramming the cell cycle machinery to treat cardiovascular disease.

Coronary artery disease is one of the most common heart pathologies. Restriction of blood flow to the heart by atherosclerotic lesions, leading to angina pectoris and myocardial infarction, damages the heart, resulting in impaired cardiac function. Damaged myocardium is replaced by scar tissue since surviving cardiomyocytes are unable to proliferate to replace lost heart tissue. Although narrowing of the coronary arteries can be treated successfully using coronary revascularisation procedures, re-occlusion of the treated vessels remains a significant clinical problem. Cell cycle control mechanisms are key in both the impaired cardiac repair by surviving cardiomyocytes and re-narrowing of treated vessels by maladaptive proliferation of vascular smooth muscle cells. Strategies targeting the cell cycle machinery in the heart and vasculature offer promise both for the improvement of cardiac repair following MI and the prevention of restenosis and bypass graft failure following revascularisation procedures.

Transthoracic Echocardiographic Examination in the Rabbit Model.

Large animal models such as the rabbit are valuable for translational preclinical research. Rabbits have a similar cardiac electrophysiology compared to that of humans and that of other large animal models such as dogs and pigs. However, the rabbit model has the additional advantage of lower maintenance costs compared to other large animal models. The longitudinal evaluation of cardiac function using echocardiography, when appropriately implemented, is a useful methodology for preclinical assessment of novel therapies for heart failure with reduced ejection fraction (e.g. cardiac regeneration). The correct use of this non-invasive tool requires the implementation of a standardized examination protocol following international guidelines. Here we describe, step by step, a detailed protocol supervised by veterinary cardiologists for performing echocardiography in the rabbit model, and demonstrate how to correctly obtain the different echocardiographic views and imaging planes, as well as the different imaging modes available in a clinical echocardiography system routinely used in human and veterinary patients.

Nox2 contributes to age-related oxidative damage to neurons and the cerebral vasculature.

Oxidative stress plays an important role in aging-related neurodegeneration. This study used littermates of WT and Nox2-knockout (Nox2KO) mice plus endothelial cell-specific human Nox2 overexpression-transgenic (HuNox2Tg) mice to investigate Nox2-derived ROS in brain aging. Compared with young WT mice (3-4 months), aging WT mice (20-22 months) had obvious metabolic disorders and loss of locomotor activity. Aging WT brains had high levels of angiotensin II (Ang II) and ROS production; activation of ERK1/2, p53, and γH2AX; and losses of capillaries and neurons. However, these abnormalities were markedly reduced in aging Nox2KO brains. HuNox2Tg brains at middle age (11-12 months) already had high levels of ROS production and activation of stress signaling pathways similar to those found in aging WT brains. The mechanism of Ang II-induced endothelial Nox2 activation in capillary damage was examined using primary brain microvascular endothelial cells. The clinical significance of Nox2-derived ROS in aging-related loss of cerebral capillaries and neurons was investigated using postmortem midbrain tissues of young (25-38 years) and elderly (61-85 years) adults. In conclusion, Nox2 activation is an important mechanism in aging-related cerebral capillary rarefaction and reduced brain function, with the possibility of a key role for endothelial cells.

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling.

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine-threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

Developmental expression of myostatin in cardiomyocytes and its effect on foetal and neonatal rat cardiomyocyte proliferation.

OBJECTIVES: Myostatin, a member of the transforming growth factor-beta (TGF-beta) family, plays a key role in skeletal muscle myogenesis by limiting hyperplastic and hypertrophic muscle growth. In cardiac muscle, myostatin has been shown to limit agonist-induced cardiac hypertrophic growth. However, its role in cardiac hyperplastic growth remains undetermined. The aim of this study was to characterise the expression of myostatin in developing myocardium, determine its effect on cardiomyocyte proliferation, and explore the signalling mechanisms affected by myostatin in dividing cardiomyocytes. METHODS: We used quantitative PCR and Western blotting to study the expression of myostatin in cardiomyocytes isolated from rat myocardium at different developmental ages. We determined the effect of recombinant myostatin on proliferation and cell viability in dividing cardiomyocytes in culture. We analysed myostatin's effect on cardiomyocyte cell cycle progression by flow cytometry and used Western blotting to explore the signalling mechanisms involved. RESULTS: Myostatin is expressed differentially in cardiomyocytes during cardiac development such that increasing expression correlated with a low cardiomyocyte proliferation index. Proliferating foetal cardiomyocytes, from embryos at 18 days of gestation, expressed low levels of myostatin mRNA and protein, whereas isolated cardiomyocytes from postnatal day 10 hearts, wherein the majority of cardiomyocytes have lost their ability to proliferate, displayed a 6-fold increase in myostatin expression. Our in vitro studies demonstrated that myostatin inhibited proliferation of dividing foetal and neonatal cardiomyocytes. Flow cytometric analysis showed that this inhibition occurs mainly via a block in the G1-S phase transition of the cardiomyocyte cell cycle. Western blot analysis showed that part of the mechanism underpinning the inhibition of cardiomyocyte proliferation by myostatin involves phosphorylation of SMAD2 and altered expressions of the cell cycle proteins p21 and CDK2. CONCLUSIONS: We conclude that myostatin is an inhibitor of cardiomyocyte proliferation with the potential to limit cardiomyocyte hyperplastic growth by altering cardiac cell cycle progression.

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model.

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Nox2 regulates endothelial cell cycle arrest and apoptosis via p21cip1 and p53.

Endothelial cells (EC) express constitutively two major isoforms (Nox2 and Nox4) of the catalytic subunit of NADPH oxidase, which is a major source of endothelial reactive oxygen species. However, the individual roles of these Noxes in endothelial function remain unclear. We have investigated the role of Nox2 in nutrient deprivation-induced cell cycle arrest and apoptosis. In proliferating human dermal microvascular EC, Nox2 mRNA expression was low relative to Nox4 (Nox2:Nox4 approximately 1:13), but was upregulated 24 h after starvation and increased to 8+/-3.5-fold at 36 h of starvation. Accompanying the upregulation of Nox2, there was a 2.28+/-0.18-fold increase in O2.- production, a dramatic induction of p21cip1 and p53, cell cycle arrest, and the onset of apoptosis (all p<0.05). All these changes were inhibited significantly by in vitro deletion of Nox2 expression and in coronary microvascular EC isolated from Nox2 knockout mice. In Nox2 knockout cells, although there was a 3.8+/-0.5-fold increase in Nox4 mRNA expression after 36 h of starvation (p<0.01), neither O2.- production nor the p21cip1 or p53 expression was increased significantly and only 0.46% of cells were apoptotic. In conclusion, Nox2-derived O2.-, through the modulation of p21cip1 and p53 expression, participates in endothelial cell cycle regulation and apoptosis.

Characterisation of cyclin D1 down-regulation in coronavirus infected cells.

The positive strand RNA coronavirus, infectious bronchitis virus (IBV), induces a G2/M phase arrest and reduction in the G1 and G1/S phase transition regulator cyclin D1. Quantitative real-time RT-PCR and Western blot analysis demonstrated that cyclin D1 was reduced post-transcriptionally within infected cells independently of the cell-cycle stage at the time of infection. Confocal microscopy revealed that cyclin D1 decreased in IBV-infected cells as infection progressed and inhibition studies indicated that a population of cyclin D1 could be targeted for degradation by a virus mediated pathway. In contrast to the SARS-coronavirus, IBV nucleocapsid protein did not interact with cyclin D1.

The use of proteomics to identify novel therapeutic targets for the treatment of disease.

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Aging-associated metabolic disorder induces Nox2 activation and oxidative damage of endothelial function.

Oxidative stress attributable to the activation of a Nox2-containing NADPH oxidase is involved in the development of vascular diseases and in aging. However, the mechanism of Nox2 activation in normal aging remains unclear. In this study, we used age-matched wild-type (WT) and Nox2 knockout (KO) mice at 3-4 months (young); 11-12 months (middle-aged) and 21-22 months (aging) to investigate age-related metabolic disorders, Nox2 activation and endothelial dysfunction. Compared to young mice, middle-aged and aging WT mice had significant hyperglycaemia, hyperinsulinaemia, increased systemic oxidative stress and higher blood pressure. Endothelium-dependent vessel relaxation to acetylcholine was significantly impaired in WT aging aortas, and this was accompanied by increased Nox2 and ICAM-1 expressions, MAPK activation and decreased insulin receptor expression and signaling. However, these aging-associated disorders were significantly reduced or absent in Nox2KO aging mice. The effect of metabolic disorder on Nox2 activation and endothelial dysfunction was further confirmed using high-fat diet-induced obesity and insulin resistance in middle-aged WT mice treated with apocynin (a Nox2 inhibitor). In vitro experiments showed that in response to high glucose plus high insulin challenge, WT coronary microvascular endothelial cells increased significantly the levels of Nox2 expression, activation of stress signaling pathways and the cells were senescent, e.g. increased p53 and ß-galactosidase activity. However, these changes were absent in Nox2KO cells. In conclusion, Nox2 activation in response to aging-associated hyperglycaemia and hyperinsulinaemia plays a key role in the oxidative damage of vascular function. Inhibition or knockout of Nox2 preserves endothelial function and improves global metabolism in old age.

Deoxyribonuclease 1 reduces pathogenic effects of cigarette smoke exposure in the lung.

Our aim was to investigate if deoxyribonuclease (DNase) 1 is a potential therapeutic agent to reduce pathogenic effects of cigarette smoke exposure in the lung. Cigarette smoke causes protease imbalance with excess production of proteases, which is a key process in the pathogenesis of emphysema. The mechanisms responsible for this effect are not well-defined. Our studies demonstrate both in vitro and in vivo that cigarette smoke significantly increases the expression of neutrophil and macrophage extracellular traps with coexpression of the pathogenic proteases, neutrophil elastase and matrix metalloproteinases 9 and 12. This response to cigarette smoke was significantly reduced by the addition of DNase 1, which also significantly decreased macrophage numbers and lung proteolysis. DNase 1, a treatment currently in clinical use, can diminish the pathogenic effects of cigarette smoke.

Characterisation and regulation of E2F-6 and E2F-6b in the rat heart: a potential target for myocardial regeneration?

The E2F transcription factors are instrumental in regulating cell cycle progression and growth, including that in cardiomyocytes, which exit the cell cycle shortly after birth. E2F-6 has been demonstrated to act as a transcriptional repressor; however, its potential role in normal cardiomyocyte proliferation and hypertrophy has not previously been investigated. Here we report the isolation and characterisation of E2F-6 and E2F-6b in rat cardiomyocytes and consider its potential as a target for myocardial regeneration following injury. At the mRNA level, both rat E2F-6 and the alternatively spliced variant, E2F-6b, were expressed in E18 myocytes and levels were maintained throughout development into adulthood. Interestingly, E2F-6 protein expression was down-regulated during myocyte development suggesting that it is regulated post-transcriptionally in these cells. During myocyte hypertrophy, the mRNA expressions of E2F-6 and E2F-6b were not regulated whereas E2F-6 protein was up-regulated significantly. Indeed, E2F-6 protein expression levels closely parallel the developmental withdrawal of myocytes from the cell cycle and the subsequent reactivation of their cell cycle machinery during hypertrophic growth. Furthermore, depletion of E2F-6, using anti-sense technology, results in death of cultured neonatal myocytes. Taken together, abrogation of E2F-6 expression in neonatal cardiomyocytes leads to a significant decrease in their viability, consistent with the notion that E2F-6 might be required for maintaining normal myocyte growth.

IL6 Trans-signaling Promotes KRAS-Driven Lung Carcinogenesis.

Oncogenic KRAS mutations occur frequently in lung adenocarcinoma. The signaling pathways activated by IL6 promote Kras-driven lung tumorigenesis, but the basis for this cooperation is uncertain. In this study, we used the gp130(F/F) (Il6st) knock-in mouse model to examine the pathogenic contribution of hyperactivation of the STAT3 arm of IL6 signaling on KRAS-driven lung tumorigenesis. Malignant growths in the gp130(F/F):Kras(G12D) model displayed features of atypical adenomatous hyperplasia, adenocarcinoma in situ, and invasive adenocarcinoma throughout the lung, as compared with parental Kras(G12D) mice, where STAT3 was not hyperactivated. Among IL6 family cytokines, only IL6 was upregulated in the lung. Accordingly, normalization of pulmonary STAT3 activity, by genetic ablation of either Il6 or Stat3, suppressed the extent of lung cancer in the model. Mechanistic investigations revealed elevation in the lung of soluble IL6 receptor (sIL6R), the key driver of IL6 trans-signaling, and blocking this mechanism via interventions with an anti-IL6R antibody or the inhibitor sgp130Fc ameliorated lung cancer pathogenesis. Clinically, expression of IL6 and sIL6R was increased significantly in human specimens of lung adenocarcinoma or patient serum. Our results offer a preclinical rationale to clinically evaluate IL6 trans-signaling as a therapeutic target for the treatment of KRAS-driven lung adenocarcinoma.

Cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors: detection methods and activity measurements.

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both cyclin/Cdk activity in cells and the extent of CDKI inhibitory activity present in a particular cell type.