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Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxiainducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-let on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/H1F-1[alpha]/herpes simplex virus protein VP 16 and Ad2/HIF-1[alpha]/nuclear factor-[kappa]B (NF-[kappa]B)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1[alpha]VP16 and Ad2/ HIF-1[alpha]/NF-[kappa]B mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1[alpha] protein levels. These results suggest that adenovirus-mediated expression of HIF-1[alpha]/VP16 or HIF-1[alpha]/NF-[kappa]B, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes. ischemic preconditioning: myocardial protection

Expression of angiopoietins in renal epithelial and clear cell carcinoma cells: regulation by hypoxia and participation in angiogenesis. Am J Physiol Renal Physiol 287: F649-F657, 2004. First published June 15, 2004; 10.1152/ajprenal.00028.2004.--The hereditary von Hippel-Lindau (VHL) syndrome predisposes sufferers to highly vascularized tumors such as renal clear cell carcinoma (RCC) and central nervous system hemangioblastoma. In RCC4 and RCC786-0 VH[L.sup.-] ceils with VHL mutations, the protein of hypoxia-inducible factor-1[alpha] (HIF-1[alpha] is constitutively stabilized and the mRNA levels of HIF target genes, including vascular endothelial growth factor (VEGF), are elevated. However, the expression of angiopoietins in these cells and their involvement in angiogenesis are not well known. In this study, we compared the mRNA levels of angiopoietins in human kidney proximal tubule epithelial (RPTE) and RCC4 and RCC786-0 VH[L.sup.-] cells. In RPTE cells, angiopoietin-4 (Ang-4) expression was selectively induced by hypoxia or by expression of a hybrid form of HIF-1[alpha]. Under normoxic conditions, the mRNA levels of Ang-4 were higher in RCC4 and RCC786-0 VH[L.sup.+] than RPTE cells. Angiopoietin-1 expression was detectable in RCC4 and RCC786-0 VH[L.sup.-] cells but not RPTE cells. In RCC786-0 VH[L.sup.+] cells, which were stably transfected with a wild-type copy of VHL, the mRNA levels of VEGF and Ang-4 were suppressed and the hypoxic response was restored. We also demonstrated that stimulation of endothelial tube formation by conditioned medium harvested from RCC4 cells was inhibited by a soluble Tie-2 receptor. These results suggest that the angiopoietin/Tie-2 system may participate in the angiogenic response to hypoxia in renal tissues and in tumor angiogenesis in renal carcinoma. hypoxia-inducible factor-[alpha] ; von Hippel-Lindau; vascular endothelial growth factor; Tie-2 receptor

The role of exogenous .nitric oxide (.NO) in the modulation of heterologous gene expression in polarized kidney epithelial cells that were stably transduced with a cDNA encoding human wild-type cystic fibrosis membrane conductance regulator (CFTR) was assessed. The results showed that .NO impaired the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms. Furthermore, the evidence indicated the potential presence of large amounts of .NO in the lung.

A study was conducted to examine the ability of deoxyspergualin (DSG) to influence the trafficking of Delta-F508-cystic fibrosis transmembrane conductance regulatory (CFTR) to the cell membrane. Chloride channel activity was analyzed using the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)-quinolinium. Results indicated that the DSG treatment of human CF cells restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane.

A series of recombinant replication-defective adenovirus gene transfer vectors with different polyadenylation signal sequences were constructed with cystic fibrosis transmembrane conductance regulators (CTRF). The recombinant adenovirus gene vectors with built-in Ad2E4ORF6 backbones exhibited efficient gene transduction capabilities during cystic fibrosis gene therapy. The recombinant adenovirus gene vectors also corrected cyclic adenosine monophosphate-mediated Cl- secretion and fluid transport through gene transduction.

Overexpression of wild-type cystic fibrosis transmembrane conductance regulator (CFTR), a chloride (Cl-) channel, arrests cell growth in the G(sub 2)/M phase and increases the cell volume of monkey kidney cells. CFTR overexpression in the G551D-CFTR mutant inhibits growth slightly, and there is an increase in the number of cells in the G(sub 2)/M phase. Decrease in the threshold of Cl- channel activity in the mutant produces growth defects. CFTR is capable of downregulating its activity.

Potent derivatives of butyrates may be effective in the cure of delta F508 or cystic fibrosis (CF) mutation-induced Chlorine ion channel defects because overexpression of mutant protein causes transfers of fractions of delta F508-cystic fibrosis transmembrane regulators (CFTR) into the plasma membrane. Incubation of delta F508-CFTR expressing cells at low temperature enhances transfer and maturation of mutant CFTR proteins.

Partial correction of defective [Cl.sup.-] secretion in cystic fibrosis epithelial cells by an analog of squalamine. Am J Physiol Lung Cell Mol Physiol 281: L1164-L1172, 2001.--Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated [Cl.sup.-] transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment [Cl.sup.-] transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a [Cl.sup.-] gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell [Cl.sup.-] currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-[Cl.sup.-] solution, indicating restoration of [Cl.sup.-] secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective [Cl.sup.-] secretion. cystic fibrosis transmembrane conductance regulator; nasal potential difference; Ussing chamber; whole cell patch clamp; chloride ion

Preconditioning in cultured cardiomyocytes elevates the expression of several protective genes including Glut-4 and heat shock protein (HSP)70. Hypoxia-inducible factor-1 (HIF-1) is known to mediate the transcriptional activation of hypoxia-responsive genes. In this study, we examined the effect of adenovirus-mediated expression of constitutively stable hybrid forms of HIF-1α on cardiomyocyte viability and gene expression. Cultured neonatal rat cardiomyocytes were subjected to simulated ischemia-reperfusion with or without preinfection with recombinant adenoviral vectors [Ad2/HIF-1α/herpes simplex vires protein VP16 and Ad2/HIF-1α/nuclear factor-κB (NF- κB)]. Cellular viability and mRNA levels of several cardioprotective genes were measured. We demonstrated that infection with Ad2/HIF-1α/VP16 and Ad2/ HIF-1α/NF-κB mimicked the upregulation of the mRNA levels of vascular endothelial growth factor (VEGF), Glut-1, Glut-4, HSP70, and inducible NO synthase (iNOS) and the protection of cultured neonatal rat cardiomyocytes by late-phase preconditioning against simulated ischemia-reperfusion. The same dose of a control viral vector expressing no transgene had no effect. Preconditioning also elevated HIF-1α protein levels. These results suggest that adenovirus-mediated expression of HIF-α/VP16 or HIF-1α/NF-κB, a constitutively stable hybrid transcriptional factor, protected cultured neonatal cardiomyocytes against simulated ischemia-reperfusion injury by inducing multiple protective genes. [ABSTRACT FROM AUTHOR]

Investigates the partial correction of defective chloride secretion among cystic fibrosis epithelial cells. Increase in cell membrane halide permeability via administration of a squalamine analog; Augmentation of equivalent short-circuit current through GL-172 infusion; Capability of the analog in partially correcting the defective chloride secretion.

Presents a study regarding functional activation of the cystic fibrosis trafficking mutant deltaF508-CFTR by overexpression. Effect of C127 cell overexpression of deltaF508-CFTR; Therapeutic benefit of agents capable of elevating intracellular levels; Effect of sodium butyrate on transepithelial chloride secretion in airway epithelial cells.