Biomedical subjects

B Meyrick

Publications and source records attributed to B Meyrick.

At least 19 recordsLinked to original sources

PubMed2001

Bone morphogenetic proteins, genetics and the pathophysiology of primary pulmonary hypertension.

Several recent papers have shown that both familial primary pulmonary hypertension (FPPH) and sporadic primary pulmonary hypertension (PPH) may have a common etiology that is associated with the inheritance and/or spontaneous development of germline mutations in the bone morphogenetic protein receptor (BMPR) type II gene. Because BMPR-II is a ubiquitously expressed receptor for a family of secreted growth factors known as the bone morphogenetic proteins (BMPs), these findings suggest that BMPs play an important role in the maintenance of normal pulmonary vascular physiology. In the present commentary we discuss the implications of these findings in the context of BMP receptor biology, and relate these data to the genetics and pulmonary pathophysiology of patients with PPH.

Activin Receptors, Type I↗

PubMed2001

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium.

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S-nitroso-N-acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 microM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N-acetyl-D-penicillamine). NOS activity was assessed using a fibroblast-reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura-2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 +/- 5.9; NONOate = 23.8 +/- 4.2; control = 14.5 +/- 2.8 microM); but A23187 (3 microM)-stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 +/- 2.5; NONOate= 29.8 +/- 7.7; control = 14.5 +/- 2.5fmol cGMP/microg per 10(6) cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2- to 3-fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive-feedback regulatory action of exogenous NO.

Animals↗

PubMed2001

The pathology of pulmonary artery hypertension.

The pathologic features of primary pulmonary hypertension (PPH) are well known but its pathogenesis remains uncertain. In the first section, this chapter outlines the characteristic structural changes of PPH. The second section deals with the pathogenesis of these changes drawing on animal models of chronic pulmonary hypertension. The third section deals with phenotypic alterations in cells from the wall of the pulmonary artery of hypertensive animals. The recent identification of a germlike mutation in the BMPR2 gene in patients with PPH provides a novel opportunity to further our understanding of the pathogenesis of this disease.

Animals↗

PubMed2000

Transfection of nasal mucosa with a normal alpha1-antitrypsin gene in alpha1-antitrypsin-deficient subjects: comparison with protein therapy.

We sought to determine whether a normal alpha1-antitrypsin (AAT) gene could be expressed in respiratory epithelium and whether local expression would have antiinflammatory effects. In an unblinded study, we delivered a normal AAT gene in a plasmid-cationic liposome complex to one nostril of each of five subjects with AAT deficiency; the other, untreated nostril served as a control. AAT protein concentration in nasal lavage fluid (NALF) increased in the transfected nostril (TN), but not in the control nostril (CN), of every subject, peaking on day 5 at levels about one-third normal (baseline CN, 4.1 +/- 1.2 microg/mg of protein; baseline TN, 4.3 +/- 1.3; day 5 CN, 4.0 +/- 0.5 [p = NS versus baseline]; day 5 TN, 9.0 +/- 1.7 [p < 0.5 versus baseline]); isoelectric focusing identified the transgene-generated protein (M) in the only two patients in whom the measurement was possible. The reverse transcriptase-polymerase chain reaction (RT-PCR), performed on NALF from TN and CN of four of the five subjects, was positive for transgene message in TN in all cases and negative in NALF from CN except for one time point in one subject. Interleukin 8 (IL-8) concentrations in NALF were elevated at baseline (normal [N = 10] = 2.5 +/- 0.5 ng/mg of protein; baseline TN = 5.5 +/- 0.8, p < 0.05 versus normal) and decreased after AAT transfection (TN = 2.9 +/- 0.6, p < 0.05 versus baseline) but not in the control nostril (CN = 6.5 +/- 2.2, p = NS versus baseline). NALF samples taken from four of the patients while receiving intravenous AAT protein showed normal concentrations of AAT, but IL-8 concentrations (10.5 +/- 4.2 ng/mg of protein, p = NS versus baseline) were not decreased from baseline. We conclude that plasmid-cationic liposome delivery of a normal AAT gene to the respiratory epithelium of deficient patients produces potentially therapeutic local AAT concentrations and that AAT gene therapy, unlike AAT protein therapy, is antiinflammatory.

Administration, Intranasal↗

PubMed2000

Cell-specific differences in ET-1 system in adjacent layers of main pulmonary artery. A new source of ET-1.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes sustained constriction of the pulmonary artery and modulates normal vascular tone. Endothelial cells were thought to be the major source of ET-1, but recent studies show that vascular smooth muscle cells (SMCs) are also capable of its synthesis. We examined the ET-1 and endothelin-converting enzyme-1 (ECE-1) system in cells cultured from two adjacent layers, subendothelial (L1) and inner medial (L2), of normal sheep main pulmonary artery and the response of this system to exogenous ET-1 and transforming growth factor-beta1 (TGF-beta1). End points include assessment of preproET-1 (ppET-1) and ECE-1 gene coexpression, measurement of intracellular and released ET-1, and ECE-1 activity. RT-PCR analysis revealed that ppET-1 and ECE-1 transcripts were greater in L1 than in L2 cells. The L1 cells also synthesized (L1, 3.2 +/- 0.1; L2, 1.2 +/- 0.1 fmol/10(6) cells) and released (L1, 9.2 +/- 0.5; L2, 2.3 +/-0.1 fmol/ml) greater amounts of ET-1 than L2 cells. The L2 cells internalized exogenous ET-1 in a dose-dependent manner (EC(50) 8 nmol/l) and were more responsive to exogenous ET-1 than L1 cells, showing upregulation of both the ppET-1 and ECE genes. TGF-beta1 downregulated ET-1-stimulated ppET-1 and ECE-1 transcripts but only in L2 cells. In addition, L1 cells showed greater ECE-1 activity than L2 cells, and in both, the activity was sensitive to the metalloprotease inhibitor phosphoramidon. We conclude that the ET-1 system in L1 and L2 cells is distinct. The data suggest that the two cell types have diverse functions in the arterial wall; the L1 cells, like endothelial cells, provide a local source of ET-1; and since the L2 cells are more responsive to exogenous ET-1, they are likely to affect normal pulmonary vascular tone.

Animals↗

PubMed2000

EP(2) receptor mediates bronchodilation by PGE(2) in mice.

PGE(2) is an important cyclooxygenase product that modulates airway inflammatory and smooth muscle responses. Signal transduction is mediated by four EP receptor subtypes that cause distinct effects on cell metabolism. To determine the role of EP(2) receptor activation, we produced a mouse lacking the EP(2) receptor by targeted gene disruption. The effect of aerosolized PGE(2) and other agonists was measured using barometric plethysmography and by measurements of lung resistance in mechanically ventilated mice. Inhalation of PGE(2) inhibited methacholine responses in wild-type but not in mice lacking the EP(2) receptor [EP(2)(-/-)]. After airway constriction was induced by methacholine aerosol, PGE(2) reduced the airway constriction enhanced pause in wild-type mice (from 0.88 +/- 0.15 to 0.55 +/- 0.06) but increased it in EP(2)(-/-) mice (from 0.73 +/- 0. 08 to 1.27 +/- 0.19). Similar results were obtained in mechanically ventilated mice. These data indicate that the EP(2) receptor mediates the bronchodilation effect of PGE(2).

Administration, Inhalation↗

PubMed2000

Venous and arterial changes in pulmonary veno-occlusive disease, mitral stenosis and fibrosing mediastinitis.

The pathogenesis of pulmonary veno-occlusive disease (PVOD) is not known. The diagnosis of PVOD frequently relies on its histological changes since it is often difficult to distinguish clinically from primary pulmonary hypertension. This study carried out a systematic analysis of the pulmonary venous and arterial remodelling that occurs in PVOD (n=5) and compared these changes to two other diseases affecting the pulmonary veins, mitral stenosis (MS; n=6) and fibrosing mediastinitis (FM; n=2), using established morphometric techniques. In PVOD, pronounced intimal and adventitial thickening were noted in veins of all sizes and arterialization of veins >50 microm external diameter was found. Similar changes were evident in the arterial wall, but intimal thickening was less severe than in the veins and medial thickening was more pronounced in arteries <300 microm external diameter. Eccentric intimal fibrosis of the veins was also noted for the first time in PVOD, although this feature occurred less frequently (approximately one third) than in MS. Less pronounced structural remodelling was also encountered in the veins in cases of MS and FM. The severity of the venous changes in PVOD may aid its diagnosis and lend insight into its pathogenesis. However, the similarity of the vascular changes in each form of venous hypertension also suggests that pathology alone may not always differentiate between these disease states. The similarity of the vascular changes in the three forms of venous hypertension suggests that, as in pulmonary artery hypertension, pressure, per se, is one of the triggers to vascular remodelling.

Adult↗

PubMed1998

Regional variability in preproEndothelin-1 gene expression in sheep pulmonary artery and lung during the onset of air-induced chronic pulmonary hypertension. Participation Of arterial smooth muscle cells.

We investigated preproendothelin-1 (ppET-1) gene expression in the main and midregion pulmonary artery, and peripheral lung from control sheep and from animals during the development of the structural and functional changes of air-induced chronic pulmonary hypertension (CPH). Measurement of ET-1 in lung lymph (n = 7) at 1, 4, 8, and 12 d of continuous air embolization (CAE) showed a significant increase from day 4 compared with controls (n = 4). A semiquantitative reverse transcription PCR for ppET-1 gene expression was developed using ovine-specific primers. Control sheep showed strikingly fewer ppET-1 transcripts in the midregion (22.9+/-2.3 ng cDNA equivalents) than in the main pulmonary artery and lung (736.0+/-263.7 and 705.5+/-125.7, respectively). Smooth muscle cells (SMC) isolated from the main and midregion artery of control sheep confirmed these findings and showed higher levels of intracellular ET-1 synthesis in the main versus the midregion artery. Differences in gene expression persisted during CAE. In main pulmonary artery and lung, ppET-1 transcripts fell to < 1% of controls. However, transcripts in the midregion artery showed a gradual increase. Coincubation of SMC from the midregion with ET-1 plus TGF-beta resulted in an increase in intracellular big ET-1 and a decrease in SMC from the main artery. We conclude that SMC from the main and midregion pulmonary artery are phenotypically different and suggest that local synthesis of ET-1 and TGF-beta, and increased levels of ET-1 in lung lymph, regulate ppET-1 gene expression and synthesis in arterial SMC during the development of air-induced CPH.

Animals↗

PubMed1996

Effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mRNA in bovine pulmonary artery endothelial cells.

Generation of reactive oxygen species (ROS) is a common event in the pathogenesis of acute lung injury. Endothelial cells may be both a target and a source of the ROS. Exposure of bovine pulmonary endothelial cells (BPAEC) to lipopolysaccharide (LPS) has been shown to result in intracellular generation of both ROS and the antioxidant enzyme, mangano superoxide dismutase (MnSOD). The present study investigates whether alterations in intracellular oxidant state affect LPS-stimulated cytotoxicity and induction of MnSOD mRNA. BPAEC were pretreated with either the free radical scavenger, dimethylsulfoxide (DMSO), the xanthine oxidase inhibitor, allopurinol, or N-acetylcysteine (a cysteine derivate capable of increasing glutathione stores) prior to exposure to LPS (0.1 microgram/ml) for either 4, 8 or 18 hours. We found that pretreatment of BPAEC with DMSO blocked both LPS-induced cytotoxicity and induction of the MnSOD gene. Nuclear run-off experiments demonstrated that LPS-stimulated induction of the MnSOD mRNA occurred at the transcriptional level and that DMSO blocked this event. Pretreatment with allopurinol also prevented the cytotoxicity associated with LPS but, in contrast to DMSO, did not alter induction of MnSOD mRNA. N-acetylcysteine did not affect the LPS-stimulated cytotoxicity but resulted in an early and transient reduction in induction of the MnSOD gene. We conclude that LPS stimulates generation of intracellular ROS that regulate induction of the MnSOD gene at the transcriptional level further, we conclude that LPS-stimulated cytotoxicity involves both the xanthine oxidase pathway and perhaps intracellular generation of hydroxyl radicals. The difference in the protective effect between DMSO, NAC and allopurinol suggest that upregulation of the MnSOD gene does not contribute to LPS-induced cytotoxicity.

Acetylcysteine↗

PubMed1995

In utero placement of aortopulmonary shunts. A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs.

BACKGROUND: The development of pulmonary hypertension and its associated increased vascular reactivity is a common accompaniment of congenital heart disease with increased pulmonary blood flow. Although the morphology of the pulmonary vascular changes is well described, the mechanisms of vascular remodeling and increased reactivity remain incompletely understood. METHODS AND RESULTS: To elucidate these mechanisms, we established an accurate and reliable experimental model of pulmonary hypertension with increased pulmonary blood flow. An aortopulmonary shunt was created with an 8.0-mm expanded polytetrafluoroethylene vascular graft in 11 late-gestation fetal lambs. At 1 month of age, shunted lambs had a pulmonary-to-systemic blood flow ratio of 2.2 +/- 1.2. Compared with 11 age-matched control lambs, mean pulmonary arterial pressure (44.8 +/- 11.7 versus 16.2 +/- 2.9 mm Hg) and the ratio of pulmonary to systemic arterial pressure were significantly increased (P < .05). Pulmonary vascular resistance was not significantly increased. The pulmonary vasoconstricting response to the infusion of U46619 (a thromboxane A2 mimic) or acute alveolar hypoxia also was augmented in the shunted lambs. Morphometric analysis of the barium-filled pulmonary artery bed revealed medial hypertrophy, abnormal extension of muscle distally into the walls of the intra-acinar arteries, and increased numbers of barium-filled intra-acinar arteries. CONCLUSIONS: In utero placement of aortopulmonary shunts reproduces the aberrant hemodynamic state of children with cogenital heart disease with left-to-right shunts; postnatal pulmonary hypertension, increased pulmonary blood flow, and vascular remodeling. In addition, the lambs have a unique paradoxical increase in pulmonary vascular volume that attenuates an increase in pulmonary vascular resistance. This experimental preparation provides a useful and consistent model for the study of the pathogenesis of pulmonary hypertension.

Animals↗

PubMed1995

Transforming growth factor-beta receptor expression on endothelial cells: heterogeneity of type III receptor expression.

Recent studies of whole animal responses have defined a role for circulating TGF-beta in the preservation and stabilization of microvascular endothelial function (Lefer et al. [1993] Proc. Natl. Acad. Sci. U.S.A., 90:1018-1022; Pfister et al. [1992] J. Exp. Med., 176:265-269). In order to determine which TGF-beta receptor types are responsible for this endothelial cell responsiveness, we used an affinity-labeling technique with 125I-TGF-beta 1 and -beta 2 to characterize TGF-beta receptors on five different endothelial cell cultures: early passage bovine lung and rat epididymal fat pad microvascular endothelial cells (BLMEC and REEC), established endothelial cell lines from bovine adrenal medulla capillaries (EJG), fetal bovine heart (FBHE), and bovine pulmonary artery (CPAE). Since it is known that endothelial cells from different parts of the vasculature vary with respect to cell surface antigen expression (McCarthy et al. [1991] Trends Pharmacol. Sci., 12:462-467; Augustin et al. [1994] Bioessays, 16:901-906), it is important to compare TGF-beta receptor expression on microvascular and macrovascular endothelial cells. We observed 85 kDa and 200-400 kDa labeled receptor bands and analyzed their relationship to the cloned Type II and III receptors using peptide antibodies. We used dithiothreitol and phosphoinositol-phospholipase C pretreatments to establish whether the 65 kDa labeled band which we observed corresponded to the Type I receptor or a glycophosphotidylinositol-linked binding protein. The results demonstrated that microvascular but not macrovascular endothelial cells express high levels of the Type III receptor. This differential expression of the Type III receptor indicates that distinct anatomical segments of the vasculature have distinct TGF-beta receptor profiles. The presence of the Type III receptor on micro- but not macrovascular endothelial cells may account for the reportedly different potency of TGF-beta 1 and TGF-beta 2 on these two endothelial cell types. Analysis of the 85 kDa and 65 kDa affinity-labeled bands revealed that all the endothelial cells express the Type II receptor and a band consistent with the presence of a dithiothreitol-sensitive Type I receptor. Two isoform-specific phosphoinositol-phospholipase C releasable TGF-beta binding proteins were also detected: a 60 kDa protein on one micro- (EJG) and one macro- (FBHE) vascular endothelial cell line and a 150/180 kDa protein on the macrovascular cell lines (FBHE and CPAE). These studies emphasize the heterogeneous nature of endothelial cells and underline the importance of using microvascular endothelial cells when examining TGF-beta responses related to microvascular function.

Adrenal Medulla↗

PubMed1995

Response of cultured human pulmonary artery endothelial cells to endotoxin.

Endotoxemia is the leading cause of the adult respiratory distress syndrome. The effects of endotoxin on pulmonary endothelium, both in vivo and in culture, are diverse and complicated, and vary between species and cellular origin. Species such as sheep and cows are particularly sensitive to endotoxin, whereas rats and mice are more resistant. Studies using cultured pulmonary endothelial cells confirm these findings. Such species variations lead us to question whether human pulmonary artery endothelial cells (HPAEC) are directly affected by endotoxin. The present study examined the effects of endotoxin on HPAEC. Cells were exposed to endotoxin (0.001-10 micrograms/ml) for 24 h and were examined by phase-contrast microscopy, and measurements were made of lactate dehydrogenase, prostacyclin, and prostaglandin E2 release in the cell-free supernatant. In the presence of serum, endotoxin doses as small as 0.01 microgram/ml resulted in endothelial retraction and pyknosis compared with controls (P < 0.05). Exposure to 10 micrograms/ml of endotoxin resulted in a significant increase in the number of pyknotic cells (P < 0.05), and lactate dehydrogenase release paralleled this finding. Endotoxin also resulted in a gradual increase in prostaglandin E2 release, reaching significance at 1 and 10 micrograms/ml of endotoxin (P < 0.05). A similar trend was noted for prostacyclin release. We conclude that the direct cytotoxic effects elicited by endotoxin on HPAEC may contribute to the onset of pulmonary edema in patients with adult respiratory distress syndrome.

Cells, Cultured↗

PubMed1995

Secretory leukoprotease inhibitor attenuates lung injury induced by continuous air embolization into sheep.

Continuous air embolization (CAE) into the pulmonary arterial circulation of sheep results in functional and structural changes of chronic pulmonary hypertension. Release of elastin peptides into lung lymph during CAE and attenuation of CAE-induced pulmonary hypertension by neutrophil depletion suggest that neutrophil elastase may contribute to these changes. To investigate this notion, we treated awake sheep with a potent neutrophil elastase inhibitor, recombinant secretory leukoprotease inhibitor (SLPI) (100 mg/day by aerosol), during 12 days of CAE (CAE+SLPI; n = 7). Controls included sheep receiving CAE + vehicle (VEH) (n = 6), VEH alone (n = 3), and SLPI alone (n = 3). SLPI significantly attenuated the CAE-induced increases in lung lymph flow (day 8; 2.3 +/- 0.5 vs. 5.6 +/- 1.7 ml/15 min), protein clearance (day 8; 1.36 +/- 0.32 vs. 3.08 +/- 0.84 ml/15 min), and elastin peptide concentration (day 8; 243 +/- 41 vs. 398 +/- 44 ng/ml). SLPI delayed the onset of sustained pulmonary hypertension from day 8 to day 12. Both CAE groups showed similar structural changes in the pulmonary arteries. SLPI was well tolerated in control sheep and did not affect hemodynamics or structure. We conclude that serine proteases may contribute to the early initiation of chronic pulmonary hypertension but do not play a striking role in its eventual development.

Aerosols↗

PubMed1995

Pulmonary artery adventitial changes and venous involvement in primary pulmonary hypertension.

Primary pulmonary hypertension (PPH) is associated with a spectrum of structural changes in the pulmonary arteries: increased medial thickness, eccentric and concentric intimal thickening, obliteration and recanalization of arteries, and appearance of plexiform and dilatation lesions. The purpose of the present study was to further characterize these structural changes with particular emphasis on arterial adventitial thickness and alterations in the walls of the pulmonary veins. In addition, to determine whether the characteristic structural changes of PPH were size related, each was related to external diameter. With quantitative techniques, the pulmonary vasculature of 19 patients with PPH and 7 controls was examined by light microscopy. In all 19 patients, we found a striking increase in adventitial, as well as intimal and medial, thickness in arteries of all sizes when compared with controls (P < 0.05). In addition, we found intimal and adventitial thickening of pulmonary veins < 250 mu in diameter in approximately half of the PPH cases (P < 0.05). The frequency of arterial obliteration, concentric intimal thickening, and recanalization was 16, 18, and 11 of 19 cases, respectively. These changes were most prevalent in arteries less than 200 mu in diameter whereas eccentric intimal thickening and plexiform lesions occurred in 15 and 6 of the patients, respectively, and were most widespread in arteries > 200 mu. We conclude that remodeling of the pulmonary vasculature in PPH routinely includes thickening of the arterial adventitia and frequently also includes changes in the walls of the pulmonary veins. The finding that recanalization occurs predominantly in the smaller arteries whereas eccentric intimal thickening occurs mainly in the larger ones suggests that recanalization should not be considered a consequence of thromboemboli but may also occur at sites of more fibrotic intimal change.

Adolescent↗

PubMed1994

Oxidants induce transcriptional activation of manganese superoxide dismutase in glomerular cells.

Cultured rat glomerular mesangial and epithelial cells and bovine glomerular endothelial cells were exposed to various concentrations of hydrogen peroxide (H2O2). Mesangial cells treated with 10 to 100 microM H2O2 for 24 hours showed a two- to ninefold increase in Mn-SOD mRNA expression associated with significantly (P < 0.005) increased Mn-SOD activity (22.2 +/- 1.2 and 12.2 +/- 0.7 mu/mg protein for H2O2 100 microM treated and untreated cells, respectively). In contrast, expression of Cu-Zn SOD and beta-actin mRNA was not affected by H2O2. Induction of Mn-SOD mRNA by H2O2 was inhibited by actinomycin-D (4 microM) treatment. Glomerular endothelial cells also showed an increase in Mn-SOD mRNA expression following 100 microM H2O2 treatment, as did glomerular epithelial cells following treatment with 500 and 1000 microM H2O2 but not with 100 microM. Transcriptional activity of the Mn-SOD gene was assessed with a fusion reporter gene consisting of a luciferase gene (pGL2P) and a 1.2 kb fragment from the rat Mn-SOD genomic DNA (-806 to +408 bp of the transcription initiation site, -806:+408). The construct was transfected into rat glomerular mesangial and epithelial cells. Mesangial and epithelial cells transfected with pGL2P (-806:+408) and treated with H2O2 (100 microM and 1 mM for mesangial and epithelial cells, respectively) demonstrated some threefold increase in luciferase activity, whereas cells transfected with pGL2P lacking the Mn-SOD fragment did not show changes in luciferase activity following H2O2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Identification and functional characterization of the bovine manganous superoxide dismutase promoter.

Manganous superoxide dismutase (MnSOD) gene expression is stimulated by endotoxin, tumor necrosis factor, and interleukin-1, agents thought to cause cellular damage through intracellular generation of reactive oxygen species. To study the molecular mechanisms underlying the induction of MnSOD mRNA by these stimuli, we cloned a bovine MnSOD cDNA and used it to isolate the promoter region of the bovine MnSOD gene. A 14 kb genomic DNA fragment (lambda BS1) containing the first and second exons and 5' flanking region of the gene was characterized. The transcription start site was determined by primer extension and S1 nuclease protection assays and found to be 88 bp upstream of the translation initiation codon. The sequence of approximately 1 kb of DNA upstream of the start site was determined and examined for potential regulatory elements. DNA immediately upstream of the transcription start site was GC-rich and contained two AP-2 and eight Sp-1 consensus sequences. It did not contain either a CCAAT or TATA box. A 956 bp fragment of this DNA fragment was transcriptionally active when fused to a luciferase reporter gene and transfected into both bovine pulmonary artery endothelial and hamster insulinoma tumor cells. Transfection analysis of three additional deletion mutants, whose 5' end-points were -317, -182, and -70 bp, respectively, showed a step-like reduction in transfection efficiency, suggesting the presence of regulatory elements throughout this DNA fragment that contribute to transcriptional activity of the MnSOD promoter. Despite the high homology of the bovine MnSOD cDNA to other mammalian MnSODs, the promoter sequences of bovine and rat MnSOD genes showed a virtual lack of similarity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Expression of transforming growth factor-beta mRNAs and proteins in pulmonary vascular remodeling in the sheep air embolization model of pulmonary hypertension.

Transforming growth factor-beta (TGF-beta) has been suggested as one of the mediators of vascular remodeling in chronic pulmonary hypertension. We have previously shown a transient early increase in TGF-beta levels in lung lymph during the development of sustained pulmonary hypertension in a sheep model (12 days of air embolization). The present study examines expression and cellular localization of mRNA and protein of the three mammalian isoforms of TGF-beta in lung biopsy tissue taken during the development of pulmonary hypertension (0, 1, 4, 8, and 12 days of embolization). In control tissue, immunohistochemical techniques localized each of the TGF-beta proteins in an identical pattern in large preacinar airways--bronchial epithelium and subepithelial cells--and in the medial wall of muscular vessels; no protein was detected in intraacinar regions. Following air embolization, immunoreactivity appeared in peripheral lung. At day 1, immunoreactivity for TGF-beta 1 and TGF-beta 3 proteins was seen in edema fluid, in perivascular cells associated with nonmuscular intraacinar arteries, and in alveolar walls; no increased immunoreactivity was detected for TGF-beta 2. After 4, 8, and 12 days of embolization, immunoreactivity for all three TGF-beta proteins was associated with newly muscularized intraacinar arteries. With in situ hybridization, the three TGF-beta mRNAs co-localized in lung tissue from both control and air-embolized animals. In control tissue, hybridization was seen around preacinar airways and muscular vessels; no hybridization seen in intraacinar regions of the lung. After 1 day of embolization, the pattern of hybridization was similar to controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Expression of human growth hormone fusion genes in cultured lung endothelial cells and in the lungs of mice.

We sought to develop genetic therapy for acute lung diseases by introducing genes into lung cells in vivo that were only transiently expressed. To that end, we introduced a gene encoding a physiologically relevant secreted human protein into bovine lung endothelial cells in culture and into the lungs of mice using the technique of lipofection. We exposed cultured endothelial cells to a plasmid containing the coding region for human growth hormone (hGH) driven by a metallothionein (MT) promoter. In cells lipofected with the plasmid containing the MT promoter, expression of the hGH gene in medium was low (peak = 30 ng hGH/24 h/60-mm dish), but expression was markedly increased by addition of either dexamethasone (peak = 91) or cadmium (peak = 120). Lipofection with the same construct except a thymidine kinase promoter showed no cadmium response. We gave mice 5,000 ppm ZnSO4 in their drinking water and 24 h later injected intravenously plasmid containing the MT promoter complexed to liposomes. Mice were killed 1, 3, and 5 days after injection, and hGH production by minced lung, liver, and kidneys was determined in vitro. Neither kidneys nor liver produced detectable hGH. However, hGH was produced by the lungs, beginning on day 1, peaking on day 3 (approximately 1.0 ng hGH/24 h/g tissue), and declining by day 5. Lungs from mice injected either with DNA alone or with liposome alone did not produce hGH. mRNA specific for hGH was demonstrated in the lungs by polymerase chain reaction amplification of cDNA followed by agarose gel electrophoreses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗