h-89 and Ischemia

Recently it has been demonstrated that catecholamines are produced and used by macrophages and mediate immune response. The aim of this study is to verify whether endothelial cells (ECs), which are of myeloid origin, can produce catecholamines. We demonstrated that genes coding for tyrosine hydroxylase, Dopa decarboxylase, dopamine β hydroxylase (DβH), and phenylethanolamine-N-methyl transferase, enzymes involved in the synthesis of catecholamines, are all expressed in basal conditions in bovine aorta ECs, and their expression is enhanced in response to hypoxia. Moreover, hypoxia enhances catecholamine release. To evaluate the signal transduction pathway that regulates catecholamine synthesis in ECs, we overexpressed in bovine aorta ECs either protein kinase A (PKA) or the transcription factor cAMP response element binding, because PKA/cAMP response element binding activation induces tyrosine hydroxylase transcription and activity in response to stress. Both cAMP response element binding and PKA overexpression enhance DβH and phenylethanolamine-N-methyl transferase gene expression and catecholamine release, whereas H89, inhibitor of PKA, exerts the opposite effect, evidencing the role of PKA/cAMP response element binding transduction pathway in the regulation of catecholamine release in bovine aorta ECs. We then evaluated by immunohistochemistry the expression of tyrosine hydroxylase, Dopa decarboxylase, DβH, and phenylethanolamine-N-methyl transferase in femoral arteries from hindlimbs of C57Bl/6 mice 3 days after removal of the common femoral artery to induce chronic ischemia. Ischemia evokes tyrosine hydroxylase, Dopa decarboxylase, DβH, and phenylethanolamine-N-methyl transferase expression in the endothelium. Finally, the pharmacological inhibition of catecholamine release by fusaric acid, an inhibitor of DβH, reduces the ability of ECs to form network-like structures on Matrigel matrix. In conclusion, our study demonstrates for the first time that ECs are able to synthesize and release catecholamines in response to ischemia.

Topics: Adrenergic beta-Antagonists; Animals; Blotting, Western; Catecholamines; Cattle; Cell Hypoxia; Cells, Cultured; Cyclic AMP Response Element-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Dopa Decarboxylase; Dopamine beta-Hydroxylase; Endothelial Cells; Endothelium, Vascular; Femoral Artery; Hindlimb; Immunohistochemistry; Ischemia; Isoquinolines; Mice; Mice, Inbred C57BL; Propanolamines; Receptors, Adrenergic, beta-2; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfonamides; Tyrosine 3-Monooxygenase

We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.

Topics: Animals; Carbon Monoxide; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Electron Transport Complex IV; Hypoxia; Immunoblotting; Immunoprecipitation; Ischemia; Isoquinolines; Macrophages; Male; Mice; Mitochondria; Monocytes; Myocardial Ischemia; Myocardium; Oxidative Stress; Oxygen; Peptides; Perfusion; Phosphorylation; Protein Kinase C; Rabbits; Reactive Oxygen Species; Reperfusion Injury; Sulfonamides; Time Factors

Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia, but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore, we investigated the effects of risk factors, such as glucose and TNF-alpha, on the number of EPCs in vitro to elucidate the underlying mechanisms.. EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; P<0.05, respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase, the downstream kinase mitogen- and stress-activated kinase 1, and the transcription factor cAMP-responsive element-binding protein (CREB), in EPCs. Moreover, EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; P<0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control, respectively). Likewise, ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08, P<0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64, suggesting that p38 regulates proliferation and differentiation events.. These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy.

Topics: Activating Transcription Factor 2; Adult; Aged; Amino Acid Sequence; Animals; Apoptosis; Cell Count; Cell Differentiation; Cell Division; Cells, Cultured; Comorbidity; Coronary Disease; Cyclic AMP Response Element-Binding Protein; Female; Glucose; Hindlimb; Humans; Imidazoles; Ischemia; Isoquinolines; Male; Mice; Mice, Nude; Middle Aged; Molecular Sequence Data; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Processing, Post-Translational; Pyridines; Ribosomal Protein S6 Kinases, 90-kDa; Risk Factors; Signal Transduction; Stem Cell Transplantation; Stem Cells; Sulfonamides; Transcription Factors; Transplantation, Heterologous; Tumor Necrosis Factor-alpha