Phosphodiesterase-4 promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF.

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1α and HIF2α) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Mitochondrial ageing.

Mitochondria in largely postmitotic cells (e.g. cardiomyocytes, neurons or skeletal muscle myotubes) have a limited life span of a few weeks. Their replacement during normal turnover requires an intergenomic coordination between the mitochondrial genome (mtDNA, encoding for 13 protein subunits of the respiratory chain, two mitochondrial rRNAs and the 22 mitochondrial tRNAs) and the nuclear genome (encoding for more than 99 % of the mitochondrial proteins). The mtDNA contains only a very small non-coding region, it is exposed to radicals generated by the respiratory chain during aerobic ATP formation, and mitochondrial DNA repair capacity is rather low. Therefore, oxidative damage of mtDNA, accumulating with age, should affect mitochondrially encoded proteins, but the high number of mitochondrial genomes (roughly 10 per mitochondrion) allows a certain degree of heteroplasmy (different genomes within a mitochondrion) without effects on phenotype. Therefore, age-associated increments in mtDNA damage are to a major extent an epiphenomenon. On the other hand, however, there are clonal accumulations of damaged/mutated mtDNA within individual cells up to homoplasmy of mutant mtDNA, which are either neutral with regard to phenotype or which cause substantial phenotype alterations: hyporespiratory phenotype (less radicals and less ATP!) or a phenotype with a dysproportionate respiratory chain, i.e. partial defects within the chain with enhanced radical formation proximal to this defect and with enhanced susceptibility to oxidative stress-triggered apoptosis, probably explaining the progressive loss of cardiomyocytes with advanced age. Thus, a minority of age-associated alterations in mtDNA may explain important features of the ageing heart: myocyte losses and myocyte heterogeneity. However, documentation of definite proof for this possibility is lacking and may be difficult.

Angiotensin-converting enzyme inhibitor therapy prevents upregulation of endothelin-converting enzyme-1 in failing human myocardium.

In this study, we investigated the role of the renin-angiotensin system in expression of the endothelin system in atrial myocardium of patients with congestive heart failure. Atrial myocardium of control patients without angiotensin-converting enzyme (ACE) inhibitor therapy and heart failure patients without or with ACE inhibitor therapy undergoing aorto-coronary bypass surgery was studied. Endothelin-converting enzyme-1 (ECE-1) expression and endothelin-1 peptide level was upregulated in myocardium of heart failure patients without ACE inhibition. ACE inhibitor therapy prevented upregulation of ECE-1 and endothelin-1 in failing myocardium. Prepro-endothelin-1 and endothelin receptor A expression were not affected by heart failure. Endothelin receptor B was downregulated in heart failure patients. Our data demonstrate an upregulation of ECE-1 mRNA expression in failing human myocardium. Inhibition of the renin-angiotensin system by ACE inhibitor treatment prevents upregulation of ECE-1, suggesting that angiotensin II regulates ECE-1 expression in vivo.

S-Nitrosylation of mitochondrial caspases.

Caspase-3 is a cysteine protease located in both the cytoplasm and mitochondrial intermembrane space that is a central effector of many apoptotic pathways. In resting cells, a subset of caspase-3 zymogens is S-nitrosylated at the active site cysteine, inhibiting enzyme activity. During Fas-induced apoptosis, caspases are denitrosylated, allowing the catalytic site to function. In the current studies, we sought to identify the subpopulation of caspases that is regulated by S-nitrosylation. We report that the majority of mitochondrial, but not cytoplasmic, caspase-3 zymogens contain this inhibitory modification. In addition, the majority of mitochondrial caspase-9 is S-nitrosylated. These studies suggest that S-nitrosylation plays an important role in regulating mitochondrial caspase function and that the S-nitrosylation state of a given protein depends on its subcellular localization.

Redox control of mitochondrial functions.

Redox reactions and electron flow through the respiratory chain are the hallmarks of mitochondria. By supporting oxidative phosphorylation and metabolite transport, mitochondrial redox reactions are of central importance for cellular energy conversion. In the present review, we will discuss two other aspects of the mitochondrial redox state: (i) its control of mitochondrial Ca2+ homeostasis, and (ii) the intramitochondrial formation of reactive oxygen or nitrogen species that strongly influence electron flow of the respiratory chain.

Increased expression of endothelin-1 and inducible nitric oxide synthase isoform II in aging arteries in vivo: implications for atherosclerosis.

We here report that aging increases expression of endothelin-1 and NO synthases in the vasculature and kidney of normotensive rats in vivo. Expression of preproendothelin-1 mRNA was quantified by RT-PCR and in situ hybridization, and endothelin-1 protein was determined by radioimmunoassay/HPLC. Vascular mRNA expression of NO synthase isoforms II and III was analyzed by RT-PCR. In young animals, vascular endothelin-1 protein was differentially expressed (aorta < renal artery < carotid artery) and increased with aging in all vascular beds (P < 0.05). In the intact aorta of aged rats, mRNA expression of preproendothelin-1, "inducible" NO synthase II, and endothelial cell NO synthase III gene was up-regulated (P < 0.05). Moreover, preproendothelin-1 mRNA expression increased in glomeruli and tubulointerstitial cells (P < 0.05). To our knowledge this is the first study demonstrating local vascular up-regulation of the trophic factor endothelin under physiological conditions. Activation of vascular endothelin and NO synthases may be important, pressure-independent factors contributing to structural and functional abnormalities of age-dependent diseases, including atherosclerosis.

Deloading of the left ventricle by ventricular assist device normalizes increased expression of endothelin ET(A) receptors but not endothelin-converting enzyme-1 in patients with end-stage heart failure.

BACKGROUND: Ventricular assist devices (VAD) are implanted in patients with end-stage heart failure for bridging the time until heart transplantation, resulting in hemodynamic unloading of the failing heart, improved cardiac contractile and mitochondrial function, and reversal of cardiac hypertrophy. It is unknown whether VAD unloading may affect the cardiac endothelin (ET) system, which has been proposed as one of the putative pathomechanisms of heart failure. METHODS AND RESULTS: With the use of standard-calibrated, competitive reverse-transcription-polymerase chain reaction mRNA expression of components of the ET system was analyzed in left ventricular myocardium from nonfailing donor hearts, from failing hearts without and with ACE inhibitor therapy, and from patients with end-stage heart failure at the time of VAD implantation and 103+/-15 days after VAD implantation during removal with subsequent heart transplantation. ET receptor A (ET(A)) was markedly upregulated in failing human myocardium. This increased ET(A) expression was not affected by ACE inhibitor treatment but was normalized by VAD unloading. ET(A) expression before or after VAD implantation did not correlate with duration of VAD implantation or suppression of Pro-ANP mRNA. ET(B) mRNA expression was unaffected by heart failure or VAD. In contrast, increased ET-converting enzyme-1 mRNA and ET-1 peptide levels in failing myocardium were partially normalized by ACE inhibition but not by VAD unloading. CONCLUSIONS: We conclude that VAD implantation normalizes ET(A) expression in failing human left ventricular myocardium, probably as the result of the beneficial effects of VAD unloading.

Regulation of the endothelin system by shear stress in human endothelial cells.

In this study, the effect of shear stress on the expression of genes of the human endothelin-1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm-2 (i.e. 0.1, 1.5 or 3 N m-2) (venous and two different arterial levels of shear stress) in a cone-and-plate viscometer. Laminar shear stress transiently upregulates preproendothelin-1 (ppET-1) mRNA, reaching its maximum after 30 min (approx 1.7-fold increase). In contrast, long-term application of shear stress (24 h) causes downregulation of ppET-1 mRNA in a dose-dependent manner. Arterial levels of shear stress result in downregulation of endothelin-converting enzyme-1 isoform ECE-1a (predominating in HUVEC) to 36.2 +/- 8.5 %, and isoform ECE-1b mRNA to 72.3 +/- 1.9 % of static control level. The endothelin-1 (ET-1) release is downregulated by laminar shear stress in a dose-dependent manner. This downregulation of ppET-1 mRNA and ET-1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L-NAME, 500 microm) prevents downregulation of ppET-1 mRNA by shear stress. In contrast, increasing degrees of long-term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO- and PKC-, but not tyrosine kinase-dependent mechanism. In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETB receptor by long-term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti-arteriosclerotic potential of arterial laminar shear stress.