Jean M. Herron

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells.

The transcriptional machinery involved in the transition of an infant from intrauterine to air-breathing life is developmentally regulated, as the fetus and adult manifest differential genetic expression. The low oxygen (O2) environment of the mammalian fetus and the increase in O2 tension that occurs at birth may account for the developmentally regulated alterations in gene expression. We tested the hypothesis that hypoxia-inducible factor 1 (HIF-1) expression, an O2-sensitive transcription factor, is developmentally regulated. We found that in fetal pulmonary artery (PA) smooth muscle cells (SMC), fetal HIF-1 protein levels were O2-insensitive, whereas in adult PA SMC, hypoxia increased HIF-1 protein expression. Surprisingly, hypoxia increased HIF-1 mRNA expression in fetal, but not in adult, PA SMC. HIF-1 degradation and transcriptional activity is contingent on prolyl- and asparagyl-hydroxylases. To determine whether developmental differences in O2 sensitivity or expression of these enzymes accounts for the divergence of HIF-1 sensitivity between fetus and adult, we studied the expression of the three most well characterized prolyl-hydroxylases, PHD1, PHD2, and PHD3, and the expression of regulators of HIF-1 transcriptional activity, asparagyl-hydroxylase, factor inhibiting HIF, and the oncogenic factor, CITED2 (CREB-binding protein/p300 interacting transactivator with ED-rich tail). We found that, as in the case of HIF-1, these genes are differentially regulated in the fetus, enabling the mammalian fetus to thrive in the low O2 tension intrauterine environment even while rendering a newborn infant uniquely well adapted to respond to the acute increase in O2 tension that occurs at birth.

Acute normoxia increases fetal pulmonary artery endothelial cell cytosolic Ca2+ via Ca2+-induced Ca2+ release

To test the hypothesis that an acute increase in O2 tension increases cytosolic calcium ([Ca2+]i) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (<10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (Po2, 25 Torr). Using the calcium-sensitive dye fura-2, we demonstrated that acute normoxia (Po2 = 120 Torr) increased PAECs [Ca2+]i by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca2+]i Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)–sensitive dye, DAF - FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O2 tension had no effect on either [Ca2+]i or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca2+]i and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores.

Proteinase-free myeloperoxidase increases airway epithelial permeability in a whole trachea model.

In cystic fibrosis the bronchiectatic conducting airways have large numbers of neutrophils in their walls and in their luminal contents. The neutrophils primary granule enzyme activities of elastase and peroxidase are increased in the sputum of these patients. It has been postulated that these enzymes—together or individually—act to damage the airway epithelium. However, only peroxidase activity has consistently correlated with the degree of structural and functional airway disease in these patients with leakage of plasma protein into the airway lumen (Regelmann et al., Pediatr Pulmonol. 1995; 19: 1–9). The present study was designed to test whether human neutrophil‐derived myeloperoxidase can independently produce bronchial epithelial damage without the presence of proteases, as measured by increased permeability of the airway epithelium. Human peripheral blood neutrophils were purified, their primary granules isolated, and their peroxidase purified using affinity and ion exchange column chromatography. Activity of the proteinase‐free peroxidase was measured using a chromogenic substrate. The effect of this peroxidase on the permeability of excised rat tracheas was measured using radioactive and fluorescent‐labeled non‐ionic molecules of varying molecular weight.

Pulmonary Vascular Voltage-Sensitive K+ Channel Expression is Developmentally Regulated † 1630

Pulmonary Vascular Voltage-Sensitive K+ Channel Expression is Developmentally Regulated † 1630

NEUTROPHIL DYSFUNCTION IN AUTOIMMUNE NEUTROPENIA

The role of anti-neutrophil antibodies was investigated as a cause of functional abnormalities of circulating neutrophils in a child with persistent neutropenia and recurrent mouth sores. The patients neutrophils were isolated by isopycnic centrifugation and compared with normal adult neutrophils. Functional abnormalities in the patients PMNS included (1) decreased chemotaxis studied by under agarose method and by skin window, (2) decreased oxidative burst measured by luminol amplified chemi luminescence, and (3) decreased superoxide production measured by cytochrome C reduction assay. The ability of the patients serum to bind normal neutrophils was determined by immunofluorescence. While the patients serum bound normal PMNs, this binding did not interfere with their oxidative burst. In contrast, the patients serum did not bind to his own PMNs in vitro even though his neutrophil specific antibody was anti-NAI and his PMNs were NAI positive. Therefore, the functional abnormalities of this patients PMNs were not due to neutrophil antibody binding alone, but due to a subpopulation of dysfunctional neutrophils in circulation possibly selected out by their low affinity to the circulating autoantibodies.