Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.     

Differential effects of exercise on aortic mitochondria

Routine exercise is widely recognized as cardioprotective. Exercise induces a variety of effects within the cardiovasculature, including decreased mitochondrial damage and improved aerobic capacity. It has been generally thought that the transient increase in oxidative stress associated with exercise initiates cardioprotective processes. Somewhat paradoxically, increased oxidative stress associated with cardiovascular disease (CVD) risk factors is thought to play an important role in the promotion and development of CVD. Hence, it is possible that CVD risk factors that increase oxidative stress (e.g., hypercholesterolemia) may modulate the cardioprotective effects of exercise. In this regard, the interaction between CVD risk factors and exercise on atherosclerotic lesion development and basal oxidant load is less defined. To determine the influence of preexistent hypercholesterolemia on cardioprotective effects of exercise, atherosclerotic lesion formation, oxidant load, mitochondrial damage, protein nitration (3-nitrotyrosine levels), and mitochondrial enzyme activities were determined in aortic tissues from normocholesterolemic (C57 control) and hypercholesterolemic [apoliprotein E-deficient (apoE(-/-))] mice after 16 wk of regular exercise. In normocholesterolemic mice, regular exercise was associated with decreased mitochondrial damage and oxidant load and increased SOD2 and adenine nucleotide translocator activities. Exercise did not decrease endogenous oxidant load and mitochondrial damage in hypercholesterolemic mice and did not reduce atherosclerotic lesion development. These data are consistent with the notion that CVD risk factors associated with increased oxidative stress can alter the benefits of exercise and that mitochondrial damage appears to be correlated with the cardiovascular effects of exercise.     

Identification of a protective role for protein phosphatase 1cgamma1 against oxidative stress-induced vascular smooth muscle cell apoptosis

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1cgamma1 (catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1cgamma1 activity and expression. Depletion of PP1cgamma1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1cgamma1 overexpression abrogated DMNQ-induced JNK1 activity, p53 Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1cgamma1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1cgamma1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.     

JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.     

Heterodimer-heterotetramer formation mediates enhanced sensor activity in a biophysical model for BMP signaling

Numerous stages of organismal development rely on the cellular interpretation of gradients of secreted morphogens including members of the Bone Morphogenetic Protein (BMP) family through transmembrane receptors. Early gradients of BMPs drive dorsal/ventral patterning throughout the animal kingdom in both vertebrates and invertebrates. Growing evidence in Drosophila, zebrafish, murine and other systems suggests that BMP ligand heterodimers are the primary BMP signaling ligand, even in systems in which mixtures of BMP homodimers and heterodimers are present. Signaling by heterodimers occurs through a hetero-tetrameric receptor complex comprising of two distinct type one BMP receptors and two type II receptors. To understand the system dynamics and determine whether kinetic assembly of heterodimer-heterotetramer BMP complexes is favored, as compared to other plausible BMP ligand-receptor configurations, we developed a kinetic model for BMP tetramer formation based on current measurements for binding rates and affinities. We find that contrary to a common hypothesis, heterodimer-heterotetramer formation is not kinetically favored over the formation of homodimer-tetramer complexes under physiological conditions of receptor and ligand concentrations and therefore other mechanisms, potentially including differential kinase activities of the formed heterotetramer complexes, must be the cause of heterodimer-heterotetramer signaling primacy. Further, although BMP complex assembly favors homodimer and homomeric complex formation over a wide range of parameters, ignoring these signals and instead relying on the heterodimer improves the range of morphogen interpretation in a broad set of conditions, suggesting a performance advantage for heterodimer signaling in patterning multiple cell types in a gradient.     

Stimulation of a vascular smooth muscle cell NAD(P)H oxidase by thrombin. Evidence that p47(phox) may participate in forming this oxidase in vitro and in vivo.

Thrombin is a potent vascular smooth muscle cell (VSMC) mitogen. Because recent evidence implicates reactive oxygen intermediates (ROI) in VSMC proliferation in general and atherogenesis in particular, we investigated whether ROI generation is necessary for thrombin-induced mitogenesis. Treatment of human aortic smooth muscle cells with thrombin increased DNA synthesis, an effect that was antagonized by diphenyleneiodonium but not by other inhibitors of cellular oxidase systems. This effect of thrombin was accompanied by increased O-2 and H2O2 generation and NADH/NADPH consumption. ROI generation in response to thrombin pretreatment could also be blocked by diphenyleneiodonium, suggesting that the NAD(P)H oxidase was necessary for ROI generation and thrombin-induced mitogenesis. Because of observed differences between the VSMC and neutrophil oxidase, we examined whether the cytosolic components of the phagocytic NAD(P)H oxidase were present in VSMC. p47(phox) and Rac2 were present in VSMC. Furthermore, thrombin increased expression of p47(phox) and Rac2 and stimulated their translocation to the cell membrane. We examined whether p47(phox) might be similarly regulated in vivo in a rat aorta balloon injury model and found that p47(phox) protein was increased after injury. Immunocytochemistry localized expression of p47(phox) to the neointima and media of injured arteries. Our data demonstrate that generation of O-2 and H2O2 is required for thrombin-mediated mitogenesis in VSMC and that p47(phox) is regulated by thrombin in vitro and is associated with vascular lesion formation in vivo.     

Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway

An ergostane type triterpenoid methylantcinate A (MAA) isolated from the fruiting bodies of Antrodia camphorata inhibited the growth of oral cancer cell lines OEC-M1 and OC-2 in a dose-dependent manner, without cytotoxic to normal oral gingival fibroblast cells. The major mechanism of growth inhibition was apoptosis induction, as shown by flow cytometric analysis of annexin V-FITC and propidium iodide staining, caspase-3 activation and DNA fragmentation. The increased expression of pro-apoptotic Bax, poly-(ADP-ribose) polymerase cleavage, and activated caspase-3 and decreased expression of anti-apoptotic Bcl-2 and Bcl-xL were also observed. These results provide the first evidence that the anti-oral cancer effects of MAA may involve a mechanism through the mitochondrial dependent pathway. Thus, results reported here may offer further impulse to the development of MAA analogues as potential chemotherapeutic targets for oral cancer complications.