Progress in the development of kinase inhibitors for treating asthma and COPD.

Current therapies to mitigate inflammatory responses involved in airway remodeling and associated pathological features of asthma and chronic obstructive pulmonary disease (COPD) are limited and largely ineffective. Inflammation and the release of cytokines and growth factors activate kinase signaling pathways that mediate changes in airway mesenchymal cells such as airway smooth muscle cells and lung fibroblasts. Proliferative and secretory changes in mesenchymal cells exacerbate the inflammatory response and promote airway remodeling, which is often characterized by increased airway smooth muscle mass, airway hyperreactivity, increased mucus secretion, and lung fibrosis. Thus, inhibition of relevant kinases has been viewed as a potential therapeutic approach to mitigate the debilitating and, thus far, irreversible airway remodeling that occurs in asthma and COPD. Despite FDA approval of several kinase inhibitors for the treatment of proliferative disorders, such as cancer and inflammation associated with rheumatoid arthritis and ulcerative colitis, none of these drugs have been approved to treat asthma or COPD. This review will provide a brief overview of the role kinases play in the pathology of asthma and COPD and an update on the status of kinase inhibitors currently in clinical trials for the treatment of obstructive pulmonary disease. In addition, potential issues associated with the current kinase inhibitors, which have limited their success as therapeutic agents in treating asthma or COPD, and alternative approaches to target kinase functions will be discussed.

Kinase inhibitors in the treatment of obstructive pulmonary diseases.

Chronic pulmonary diseases, including chronic obstructive pulmonary disease (COPD) and asthma, are major causes of death and reduced quality of life. Characteristic of chronic pulmonary disease is excessive lung inflammation that occurs in response to exposure to inhaled irritants, chemicals, and allergens. Chronic inflammation leads to remodeling of the airways that includes excess mucus secretion, proliferation of smooth muscle cells, increased deposition of extracellular matrix proteins and fibrosis. Protein kinases have been implicated in mediating inflammatory signals and airway remodeling associated with reduced lung function in chronic pulmonary disease. This review will highlight the role of protein kinases in the lung during chronic inflammation and examine opportunities to use protein kinase inhibitors for the treatment of chronic pulmonary diseases.

Regulating the regulator: NF-kappaB signaling in heart.

The Nuclear Factor-kappaB (NF-kappaB) signaling pathway has been linked to several pathologic processes in the myocardium including cardiomyocyte proinflammatory cytokine release, ischemia/reperfusion injury, hypertrophy and apoptosis. However, very little is known about the intracellular mechanisms that govern NF-kappaB activity in the myocardial cells. Recent advances in our understanding of the regulation of NF-kappaB signaling in non-myocyte systems suggest that the activity of the NF-kappaB pathway is tightly regulated by a diversity of stress-activated signaling intermediates through direct post-translational modification of various components of the NF-kappaB pathway. In this review, we will focus on these recent revelations and their implications not only in cardiac pathologies, but in the development of new therapeutic strategies to manage heart disease.

TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia.

Tumor necrosis factor (TNF)-alpha is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-alpha-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-alpha displayed a dose- and time-dependent increase in transendothelial [(14)C]albumin flux in the absence of EC injury. TNF-alpha also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and beta-catenin, gamma-catenin, and p120 catenin (p120(ctn)). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-alpha effect. TNF-alpha activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against approximately 60% of the TNF-alpha-induced increment in [(14)C]albumin flux. PP2 also blocked TNF-alpha-induced tyrosine phosphorylation of VE-cadherin, gamma-catenin, and p120(ctn). To identify which src family kinase(s) was required for TNF-alpha-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-alpha effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-alpha regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.

Inhibitor-kappaB kinase-beta regulates LPS-induced TNF-alpha production in cardiac myocytes through modulation of NF-kappaB p65 subunit phosphorylation.

TNF-alpha is recognized as a significant contributor to myocardial dysfunction. Although several studies suggest that members of the NF-kappaB family of transcription factors are essential regulators of myocardial TNF-alpha gene expression, recent developments in our understanding of the modulation of NF-kappaB activity through posttranslational modification of NF-kappaB subunits suggest that the present view of NF-kappaB-dependent cytokine expression in heart is incomplete. Therefore, the goal of the present study was to examine the role of p65 subunit phosphorylation in the regulation of TNF-alpha production in cultured neonatal ventricular myocytes. Bacterial LPS-induced TNF-alpha production is accompanied by a 12-fold increase in phosphorylation of p65 at Ser536, a modification associated with enhancement of p65 transactivation potential. Pharmacological inhibition of IKK-beta reduced LPS-induced TNF-alpha production 38-fold, TNF-alpha mRNA levels 6-fold, and IkappaB-alpha phosphorylation 5-fold and degraded IkappaB-alpha 2-fold and p65 phosphorylation 6-fold. Overexpression of dominant-negative p65 reduced TNF-alpha production 3.5-fold, whereas overexpression of dominant-negative IKK-beta reduced LPS-induced TNF-alpha production 2-fold and p65 phosphorylation 2-fold. Overexpression of dominant-negative IKK-alpha had no effect on p65 phosphorylation or TNF-alpha production, revealing that IKK-beta, not IKK-alpha, plays a central role in regulation of p65 phosphorylation at Ser536 and TNF-alpha production in heart. Finally, we demonstrated, using a chromatin immunoprecipitation assay, that LPS stimulates recruitment of Ser536-phosphorylated p65 to the TNF-alpha gene promoter in cardiac myocytes. Taken together, these data provide compelling evidence for the role of NF-kappaB signaling in TNF-alpha gene expression in heart and highlight the importance of this proinflammatory gene-regulatory pathway as a potential therapeutic target in the management of cytokine-induced myocardial dysfunction.

p38 MAP kinase mediates inflammatory cytokine induction in cardiomyocytes and extracellular matrix remodeling in heart.

BACKGROUND: Increasing evidence suggests that development of heart failure involves activation of stress-response inflammatory cytokines, including tumor necrosis factor-alpha and interleukin-6. Yet, the myocyte contribution to their induction in failing hearts and the underlying regulatory mechanism in stressed myocardium remain unclear. METHODS AND RESULTS: In cultured cardiac myocytes, specific activation of stress-activated mitogen-activated protein kinase, p38, by upstream activator MKK6bE led to significant induction of tumor necrosis factor-alpha and interleukin-6 secretion, whereas treating cells with a selective p38 inhibitor (SB239068) significantly blocked the cytokine secretion from myocytes and increased their intracellular accumulation. Targeted expression of MKK6bE in transgenic hearts also resulted in a marked elevation in plasma tumor necrosis factor-alpha and interleukin-6; oral administration of SB239068 resulted in a significant reduction in their plasma levels but an increase in intracardiac accumulation of both cytokines. MKK6bE transgenic hearts developed marked interstitial fibrosis with increased matrix metalloproteinase abundance and selective induction of tissue inhibitor of matrix metalloproteinase-1; this extracellular matrix remodeling was also significantly attenuated by p38 inhibition. Along with cytokine induction and extracellular remodeling, MKK6bE transgenic animals displayed impaired hemodynamic function, whereas p38 inhibition improved the cardiac performance and prolonged the survival of the animals. CONCLUSIONS: Stress-activated p38 kinase is a critical regulator of inflammatory response in cardiomyocytes with significant contribution to pathological remodeling in stressed myocardium. Inhibition of p38 may represent a useful therapeutic avenue to ameliorate cardiac pathology and heart failure evolution.

G-CSF, but not corticosterone, mediates circulating neutrophilia induced by febrile-range hyperthermia.

We previously showed that sustained exposure to febrile-range hyperthermia (FRH) for 24 h caused an increase in circulating granulocyte colony-stimulating factor (G-CSF) levels and a peripheral neutrophilia in mice (Hasday J, Garrison A, Singh I, Standiford T, Ellis G, Rao S, He JR, Rice P, Frank M, Goldblum S, and Viscardi R. Am J Pathol 162: 2005-2017, 2003). In this study, we utilized a conscious temperature-clamped mouse model to analyze the kinetics of G-CSF expression and peripheral neutrophil expansion and the contributions of FRH-induced G-CSF expression, glucocorticoid generation, and catecholamine-induced neutrophil demargination. In conscious mice housed at an ambient temperature of 34.5 degrees C, core temperature rapidly equilibrated at 39.5-40 degrees C. Peripheral neutrophil counts increased 2-fold after 24-h exposure to hyperthermia, peaked at 3.6-fold baseline levels after 36-h exposure to FRH, and returned to baseline levels after 42 h of sustained hyperthermia. Plasma G-CSF levels were increased by 6.8-fold after 24 h and peaked at 40-fold baseline levels after 36 h in the hyperthermic mice. Plasma corticosterone levels peaked at 3.3-fold baseline levels after 30-h sustained hyperthermia and returned to baseline by 42 h. Immunoneutralization of G-CSF blocked FRH-induced peripheral neutrophilia, but blockade of the glucocorticoid receptor with mifepristone failed to modify FRH-induced neutrophilia. Epinephrine induced similar increases in peripheral blood absolute neutrophil counts in euthermic mice (2.2-fold increase) and mice exposed to FRH for 36 h (1.8-fold increase). Collectively, these data suggest that FRH-induced expression of G-CSF drives the sustained peripheral neutrophilia that occurs during sustained (36 h) hyperthermia, whereas glucocorticoid generation and catecholamine-induced demargination play little role in this response.

Endotoxin stress-response in cardiomyocytes: NF-kappaB activation and tumor necrosis factor-alpha expression.

Although tumor necrosis factor (TNF)-alpha is implicated in numerous cardiac pathologies, the intracellular events leading to its production by heart cells are largely unknown. The goal of the present study was to identify the role of the transcription factor nuclear factor (NF)-kappaB in this process. Among the many inducers of TNF-alpha expression in myeloid cells, only lipopolysaccharide (LPS) led to its induction in cultured neonatal myocytes. LPS also activated the NF-kappaB pathway, as evidenced by the degradation of the inhibitory protein IkappaB and the appearance of NF-kappaB-binding complexes in nuclear extracts. Furthermore, inhibitors of NF-kappaB activation, such as lactacystin, MG132, and pyrrolidine dithiocarbamate, were found to completely block the production of TNF-alpha in response to LPS stimulation, indicating a requirement of NF-kappaB for TNF-alpha expression. However, interleukin-1beta and phorbol 12-myristate 13-acetate also activated NF-kappaB but did not evoke TNF-alpha expression, revealing that this factor is not sufficient for cytokine production. Detailed examination of the NF-kappaB cascade revealed that cardiac cells displayed a unique pattern of IkappaB degradation in response to LPS, with IkappaBbeta but not IkappaBalpha being degraded upon stimulation. Additionally, two specific p65-containing DNA-binding complexes were observed in the nuclear extracts of neonatal cardiomyocytes: an inducible complex that is necessary for TNF-alpha expression and a constitutive species. Taken together, these results reveal that NF-kappaB is not only involved in cytokine production but also may be linked to other pathways that subserve a constitutive, protective mechanism for the heart cell.