Clinical practice. Acute bronchitis.
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Acute Disease↗
Biomedical subjects
Alpha A Fowler
Publications and source records attributed to Alpha A Fowler.
Hypoxia inducible factor-1 activation by prolyl 4-hydroxylase-2 gene silencing attenuates myocardial ischemia reperfusion injury.
Hypoxia inducible factor-1 (HIF-1) regulates changes in transcription of key genes such as inducible NO synthase (iNOS) in hypoxic/ischemic environments. In normoxia, HIF-1 activation is controlled by HIF-1alpha-prolyl 4-hydroxylases, which target HIF-1alpha for ubiquitination and proteasomal degradation. We hypothesized that normoxic HIF-1 preservation could attenuate cardiac ischemia/reperfusion injury via a preconditioning effect. HIF-1 preservation was achieved by using small interfering RNA (siRNA) to silence murine HIF-1alpha-prolyl-4 hydroxylase-2 (PHD2). PHD2 siRNA reduced PHD2 mRNA expression 89+/-1.5% (P<0.001) in a time- and concentration-dependent manner in normoxic murine microvascular endothelial cells (EC). PHD2 silencing in normoxic EC stabilized HIF-1alpha protein levels while significantly increasing HIF-1 transcriptional activity and iNOS mRNA expression. Wild-type mice infused with PHD2 siRNA (1.5 microg/g body weight) showed a 61+/-2.4% (P<0.05) reduction in cardiac PHD2 mRNA within 24 hours. In addition HIF-1alpha protein levels and HIF-1-dependent iNOS mRNA levels were increased. PHD2 siRNA-transfected hearts from wild-type mice (n=6) subjected to 30 minutes ischemia followed by 60 minutes reperfusion exhibited reduced infarct size when compared with saline-treated controls (9.7+/-1.9% versus 31.6+/-1.8%, respectively, P<0.0001, n=6) and to control mice transfected with a nontargeting siRNA control (28.4+/-3.0%, P<0.0001, n=6). Hearts from iNOS knockout mice receiving PHD2 siRNA by identical injection protocol (n=6) exhibited infarct size indistinguishable from saline controls (28.7+/-1.3%). These results show that in vitro and in vivo, PHD2 silencing using a siRNA strategy produces transcriptionally active HIF-1. Normoxic activation of HIF-1 in hearts following in vivo PHD2 siRNA administration attenuates reperfusion injury via an iNOS-dependent pathway.
Animals↗
HIF-1 activation attenuates postischemic myocardial injury: role for heme oxygenase-1 in modulating microvascular chemokine generation.
The CXC chemokine IL-8, which promotes adhesion, activation, and transmigration of polymorphonuclear neutrophils (PMN), has been associated with production of tissue injury in reperfused myocardium. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric peptide that is a key regulator of genes such as heme oxygenase (HO)-1 expressed under hypoxic conditions. We hypothesized that HO-1 plays an important role in regulating proinflammatory mediator production under conditions of ischemia-reperfusion. HIF-1 was activated in the human microvascular endothelial cell line (HMEC-1) with the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). DMOG significantly attenuated cytokine-induced IL-8 promoter activity and protein secretion and cytokine-induced PMN migration across human microvascular endothelial cell line HMEC-1 monolayers. In vivo studies in a rabbit model of myocardial ischemia-reperfusion showed that rabbits pretreated with a 20 mg/kg DMOG infusion (n = 6) 24 h before study exhibited a 21.58 +/- 1.76% infarct size compared with 35.25 +/- 2.06% in saline-treated ischemia-reperfusion animals (n = 6, change in reduction = 39%; P < 0.001). In DMOG-pretreated (20 mg/kg) animals, plasma IL-8 levels at 3 h after onset of reperfusion were 405 +/- 40 pg/ml vs. 790 +/- 40 pg/ml in saline-treated ischemia-reperfusion animals (P < 0.001). DMOG pretreatment reduced myocardial myeloperoxidase activity, expressed as number of PMN per gram of myocardium, to 1.43 +/- 0.59 vs. 4.86 +/- 1.1 (P = 0.012) in saline-treated ischemia-reperfused hearts. Both in vitro and in vivo DMOG-attenuated IL-8 production was associated with robust HO-1 expression. Thus our data show that HIF-1 activation induces substantial HO-1 expression that is associated with attenuated proinflammatory chemokine production by microvascular endothelium in vitro and in vivo.
Amino Acids, Dicarboxylic↗
Hypoxia inducible factor-1: regulation by nitric oxide in posthypoxic microvascular endothelium.
Microvascular endothelial cells provide a critical regulatory interface between blood constituents and tissue. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for expression of hypoxia-dependent genes. We employed a model of hypoxia and reoxygenation (H/R) using the dermal microvascular endothelial cell line HMEC-1 to examine the effects of altered oxygen concentrations on microvascular HIF-1 expression and nitric oxide (NO) formation. Hypoxia increased inducible NO synthase (iNOS) mRNA in a time-dependent manner in HMEC-1. However, endothelial NO synthase mRNA progressively declined during hypoxia. H/R promoted significant increases in cellular nitrite levels that were significantly abrogated by the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine, di hy drochloride. Exogenous NO promoted stabilization of the alpha subunit of HIF-1 and produced functional DNA binding. Exposure of HMEC-1 to H/R resulted in previously unrecognized biphasic HIF-1alpha stabilization during reoxygenation. When the iNOS gene was silenced through the use of iNOS-specific small interfering RNA, HIF-1alpha stabilization and HIF-1 activation were dramatically diminished, suggesting that inducible NOS-derived NO is a key factor sustaining HIF-1 activation during both hypoxia and reoxygenation.
Cell Line↗
Linoleic acid induces interleukin-8 production by Crohn's human intestinal smooth muscle cells via arachidonic acid metabolites.
Previously we reported that linoleic acid (LA), but not oleic acid, caused a marked increase in the secretion of IL-8 by Crohn's human intestinal smooth muscle (HISM) cells. Antioxidants inhibited this response, implicating a role for oxidative stress and NF-kappaB, a transcription factor for IL-8 that is activated by oxidative stress. In this study, we examined two mechanisms whereby LA, the dietary precursor for arachidonic acid (AA), could increase the production of IL-8 via activation of AA pathways: 1) by generation of reactive oxygen species by the AA-pathway enzymes to activate NF-kappaB or 2) by AA metabolites. Normal and Crohn's HISM cells were exposed to LA, oxidizing solution (Ox), or oxidizing solution enriched with LA (OxLA). Exposure of cells to Ox or OxLA induced oxidative stress as determined by thiobarbituric acid reactive substances. In normal cells, Ox but not LA activated NF-kappaB as determined by transfection experiments and Western blot. In Crohn's cells, NF-kappaB was spontaneously activated and was not further activated by Ox or LA. In contrast, TNF-alpha markedly increased activation of NF-kappaB in both normal and Crohn's cells. These results indicated that LA did not increase IL-8 by activating NF-kappaB, so we evaluated the second mechanism of an effect of AA metabolites. In normal cells, OxLA, but not LA, markedly stimulated IL-8, whereas in Crohn's cells, both OxLA and LA stimulated IL-8. OxLA, also stimulated production of AA metabolites leukotriene B(4) (LTB(4)), PGE(2), and thromboxane B(2) (TXB(2)) by normal and Crohn's cells. To determine whether AA metabolites mediated the IL-8 response, cells were treated with OxLA plus indomethacin (Indo), a cyclooxygenase inhibitor, and nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor. Both Indo and NDGA blocked the IL-8 response to OxLA. To determine more specifically a role for AA metabolites, AA was used. Similar to OxLA, OxAA stimulated production of IL-8 and AA metabolites. Pinane thromboxane, a selective thromboxane synthase inhibitor and receptor blocker, inhibited OxAA stimulation of TXB(2) and IL-8 in a dose-response manner. MK886, a selective 5-lipoxygenase inhibitor, inhibited OxAA stimulation of LTB(4) and IL-8 also in a dose-response manner. Analysis of specific gene products by RT-PCR demonstrated that HISM cells expressed receptors for both thromboxane and LTB(4). We conclude that AA metabolites mediated the IL-8 response to LA in HISM cells. Both cyclooxygenase and lipoxygenase pathways were involved. LA did not increase IL-8 by activating NF-kappaB, but NF-kappaB appeared to be involved, because LA increased IL-8 only in situations where NF-kappaB was activated, either spontaneously in Crohn's cells or by Ox in normal cells. We speculate that AA metabolites increased IL-8 production by enhancing NF-kappaB-dependent transcription of IL-8.
Arachidonic Acid↗
Regulation of hypoxia inducible factor-1 by nitric oxide in contrast to hypoxia in microvascular endothelium.
Hypoxia activates the transcription factor, hypoxia inducible factor-1 (HIF-1). Besides hypoxia, HIF-1 can be activated under normoxic conditions by nitric oxide. The signal transduction pathways involved in HIF-1alpha stabilization, HIF-1 DNA binding and transactivation by NO and hypoxia in microvascular endothelium remains unknown. We report that protein phosphorylation is involved in HIF-1 activation during hypoxia and NO. The phosphatidylinositol 3-kinase (PI-3K)/Akt pathway has differential effects on HIF-1 activation by hypoxia and NO. Our data indicate that the PI-3K/Akt pathway is insufficient for HIF-1alpha induction by hypoxia. The lipid and protein phosphatase activities of PTEN also appear to be involved in regulation of HIF-1alpha by NO.
Cell Line↗
Atypical mechanism of NF-kappaB activation during reoxygenation stress in microvascular endothelium: a role for tyrosine kinases.
The transcription factor nuclear factor kappaB (NF-kappaB) regulates genes that contribute to acute inflammatory reactions in cytokine-activated endothelium. Tumor necrosis factor activates NF-kappaB through serine phosphorylation, induced by inhibitor kappaB kinases (IKK), and subsequent degradation of inhibitor kappaB (IkappaB). In contrast to cytokine stress, our studies show that oxidative stress, generated by exposure to hypoxia followed by reoxygenation (H/R), failed to activate IKK in human microvascular endothelial cells (HMEC-1). We report an alternative mechanism for NF-kappaB activation during H/R stress without IkappaBalpha degradation. This mechanism involves activation of protein tyrosine kinases (PTK) that phosphorylate IkappaBalpha with peak phosphorylation occurring after 30 min of reoxygenation. Involvement of PTK was reinforced by the demonstration that the PTK inhibitor, herbimycin A, prevented H/R-mediated NF-kappaB activation. Tyrosine phosphorylation alters the association between IkappaBalpha and NF-kappaB with sufficient intensity to allow transient NF-kappaB translocation to the cell nuclei within 45 min of onset of reoxygenation stress. Immunofluorescence imaging of NF-kappaB protein reveals it to be shuttled between the nucleus and cytoplasm within 90 min of reoxygenation. Furthermore, IkappaBalpha appears to be associated with NF-kappaB during the nucleo-cytoplasmic shuttling and is thus protected from degradation. Overall, these studies suggest that tyrosine phosphorylation of IkappaBalpha represents a proteolysis-independent mechanism of NF-kappaB activation that can be targeted for preventing H/R-mediated injury without affecting normal inflammatory responses.
Cell Hypoxia↗
Reoxygenating microvascular endothelium exhibits temporal dissociation of NF-kappaB and AP-1 activation.
Alterations of cellular redox balance in microvascular endothelium results in changes of essential cell functions. These alterations may arise, in part, due to modifications in the pattern of gene expression produced by transcription factor activation. Endothelium subjected to hypoxia/reoxygenation becomes redox imbalanced, thereby leading to activation and perhaps production of a proinflammatory state. A human dermal microvascular endothelial cell line (HMEC-1) was exposed to 6 h of hypoxia (3% O(2)) followed by return to normoxia atmospheric conditions. Reactive oxygen species (ROS) generation (dichlorofluoroscein epifluorescence) was immediate and significant following reoxygenation. Electrophoretic mobility shift assays revealed activation of the oxidant sensitive transcription factors NFkappaB and AP-1, though importantly, peak activation of each factor was separated temporally by greater than 60 min. NFkappaB activation occurred without degradation of the inhibitory protein IkappaBalpha. Reoxygenating HMEC-1 exhibited a greater than 500-fold increase in polymorphonuclear neutrophil (PMN) adhesion when compared to normoxic controls. Exposure of reoxygenating HMEC-1 to the antioxidant pyrrolidine dithiocarbamate produced complete abrogation of NFkappaB activation and the intensive PMN adhesion observed in untreated, posthypoxic HMEC-1. Though rexoygenation stress induced significant upregulation of PMN adhesion, no upregulation of interleukin-8 production was observed. Our results suggest that ROS generation occurring in endothelium following onset of reoxygenation stress signals activation of key transcription factors and that their activation takes place in a temporal fashion. The temporal feature of transcription factor activation may be key to production of a postischemic proinflammatory state.
Antioxidants↗
'My doctor said I had an abnormal chest X-ray'...
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Adult↗