Pulmonary Artery Adventitia Research Articles

Hypoxia-Induced Alterations in Protein Kinase Cζ Signaling Result in Augmented Fibroblast Proliferation

Chronic hypoxic exposure induces marked remodeling in the adventitial compartment of the pulmonary artery, which is due, at least in part, to increased proliferation of the resident fibroblast populations. We have demonstrated that hypoxia can stimulate the proliferation of adventitial fibroblasts through the activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) 1/2 pathways and that those proliferative responses are greater in fibroblasts derived from chronically hypoxic animals than those from the controls. Mitogen-activated protein kinase phosphatase 1 (MKP1), an important terminator of ERK activation, has been identified as a hypoxia-inducible gene. However, the role of MKP1 in the hypoxia-induced proliferation of fibroblasts is unknown. Since hypoxia activates the PKCζ isozyme in many cell types, we hypothesized that PKCζ will control the hypoxic proliferation of vascular fibroblasts through the regulation of MKP1, and thus ERK1/2, and that this PKCζ-mediated mechanism will be different in fibroblasts from control animals vs chronically hypoxic animals. Using fibroblasts isolated from the pulmonary artery adventitia of neonatal control and chronically hypoxic calves, we found that, hypoxia, in the absence of any comitogens, induced the activation of PKCζ. The treatment of control cells with either a myristoylated pseudosubstrate peptide inhibitor or antisense oligonucleotides of PKCζ induced a marked increase in hypoxia-induced proliferation, significant up-regulation of ERK1/2 activation, and the blockade of hypoxia-stimulated increases in MKP1 levels. In contrast, in fibroblasts from chronically hypoxic calves, PKCζ inhibition blocked hypoxia-induced proliferation as well as ERK1/2 activation. MKP1 was not up-regulated in these cells under hypoxic conditions. These results demonstrate that chronic hypoxic exposure induces changes in the downstream targets of PKCζ in vascular adventitial fibroblasts. In normal fibroblasts, PKCζ-mediated MKP1 activation acts to limit cell proliferation. However, following chronic hypoxic exposure, PKCζ-regulated activation of MKP1 is lost, resulting in fibroblasts with heightened proliferative capabilities.

Commentary

HIGHLIGHTED TOPICSPulmonary Circulation and HypoxiaCommentaryGary C. SieckGary C. SieckPublished Online:01 Feb 2005https://doi.org/10.1152/japplphysiol.01298.2004MoreSectionsPDF (23 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat Exposure to chronic hypoxia, either persistent or intermittent, induces pulmonary hypertension and vascular remodeling in most mammalian species. Specifically, extensive fibroblast proliferation in the adventitial compartment in response to chronic hypoxia is common to many species including humans. In fact, fibroblasts within the pulmonary artery adventitia proliferate earlier and to a greater extent than other vascular cells in animals chronically exposed to hypoxia. Although several studies have shown that cultured fibroblasts from the pulmonary artery adventitia proliferate in response to hypoxia, the mechanisms through which hypoxia induces this autonomous growth phenotype have not been previously elucidated.In the first featured article entitled, “Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced adventitial fibroblast proliferation,” Dr. E. Gerasimovskaya and colleagues (2) determined that hypoxia activates phosphatidylinositol 3-kinase, Akt (PI3K/Akt); mammalian target of rapamycin (mTOR/p70S6K), and extracellular signal-regulated kinase (ERK1/2) signaling pathways in adventitial fibroblasts and that these pathways play a critical role in hypoxia-induced fibroblast proliferation. Activation of the mTOR/p70S6 pathway appears to be a necessary step and a key point of convergence for the ERK 1/2 and PI3K/Akt pathways that regulate cell cycle progression under hypoxic conditions. These findings suggest that, in certain cell types, hypoxic stimulation is capable of activating both transcriptional and translational pathways that are ultimately necessary for cell proliferation.Transcription factors involved in regulating the proliferative responses of fibroblasts to hypoxia have not been previously defined. In the second featured article, entitled “Egr-1 antisense oligonucleotides inhibit hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts,” Dr. M. Banks and colleagues determined that the Egr-1 transcription factor was upregulated in the pulmonary artery adventitia under hypoxic conditions in vivo and that hypoxia upregulated Egr-1 protein expression in cultured adventitial fibroblasts. Using an Egr-1 antisense strategy, these investigators demonstrated that inhibition of Egr-1 protein expression was associated with a 50% decrease in cell proliferation. Furthermore, Egr-1 antisense oligonucleotides also inhibited such downstream mediators of cell proliferation as cyclin D and epidermal growth factor receptor.Collectively, these two studies help illustrate the mechanisms through which a highly proliferative cell type can respond to hypoxia to regulate protein translational pathways and increase expression of downstream transcription factors to create an autonomous growth phenotype. They may also help explain early adventitial changes seen in hypoxic pulmonary hypertension.REFERENCES1 Banks MF, Gerasimovskaya EV, Tucker DA, Frid MG, Carpenter TC, and Stenmark KR. Egr-1 antisense oligonucleotides inhibit hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts. J Appl Physiol 98: 732–738, 2005.Link | ISI | Google Scholar2 Gerasimovskaya EV, Tucker DA, and Stenmark KR. Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced adventitial fibroblast proliferation. J Appl Physiol 98: 722–731, 2005.Link | ISI | Google Scholar Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 98Issue 2February 2005Pages 714-714 Copyright & PermissionsCopyright © 2005 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.01298.2004History Published online 1 February 2005 Published in print 1 February 2005 Metrics

Selective expansion of fibroblast subpopulations from pulmonary artery adventitia in response to hypoxia.

Proliferation of fibroblasts contributes to the adventitial thickening observed during the development of hypoxia-induced pulmonary hypertension. However, whether all or only specific subpopulations of fibroblasts proliferate during this process is unknown. Because lung, skin, and gingiva contain multiple fibroblast subpopulations, we hypothesized that the pulmonary artery (PA) adventitia of neonatal calves is composed of multiple fibroblast subpopulations and that only selective subpopulations expand under chronic hypoxic conditions. Fibroblast subpopulations were isolated from PA adventitia of control calves using limited dilution cloning techniques. These subpopulations exhibited marked differences in morphology, actin expression, and serum-stimulated growth. Only select fibroblast subpopulations demonstrated the ability to proliferate in response to hypoxia. Fibroblast subpopulations were similarly isolated from calves exposed to hypoxia (14 days). With regard to morphology, actin expression, and serum-stimulated growth of subpopulations, there were no obvious differences in fibroblast subpopulations between the hypoxic and the control calves. However, the number of fibroblast subpopulations with about a twofold increase in hypoxia-induced DNA synthesis was significantly greater in the hypoxic calves (26%) compared with control calves (10%). We conclude that the bovine PA adventitia comprises numerous phenotypically and biochemically distinct fibroblast subpopulations and that select subpopulations expand in response to chronic hypoxia.

Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers

Background: Protease activated receptor-2 (PAR-2) is a transmembrane G protein coupled receptor preferentially activated by trypsin and tryptase. The protease activated receptors play an important role in most components of...

Increased Expression of Heme Oxygenase (HO)-1 in Alveolar Spaces and HO-2 in Alveolar Walls of Smokers

It has been suggested that oxidative stress protein heme oxygenase (HO)-1 plays a role in chronic airway diseases including chronic obstructive pulmonary disease (COPD). The inducible isoform HO-1 and the constitutive HO-2 catalyze the same reaction. Their distribution in situ was studied in lungs of 10 nonsmoking subjects, 6 healthy smokers, and 10 smokers with COPD. Paraffin-embedded sections of surgical lung specimens were immunostained with antibodies against HO-1 and HO-2. HO-1 immunoreactivity was observed mainly in alveolar macrophages. HO-1-positive macrophages were increased in smokers with COPD (median: 36%) as compared with nonsmoking subjects (13%; p < 0.02), whereas no differences were observed between patients with COPD and healthy smokers (34%). HO-2 had a more widespread distribution in cells of the alveolar wall, in adventitia of pulmonary arteries and bronchioles, and in vascular smooth muscle. Lower percentages of alveolar macrophages exhibited positive staining for HO-2 without significant differences between the three groups. HO-2(+) cells in the alveolar wall were increased in smokers with (15/mm) and without COPD (12/mm) as compared with nonsmokers (8/mm, p < 0.01). In conclusion, inducible HO-1 and constitutive HO-2 are detectable in human lung tissue and their expression is increased in smokers, suggesting that oxidative stress due to cigarette smoke may increase lung cells expressing HO-1 and HO-2.

Hypoxia Induces Non-Ligand–Dependent Activation of Mitogen-Activated Protein Kinases in Pulmonary Artery Adventitial Fibroblasts: Role in Proliferation and Apoptosis

Dramatic fibroproliferative changes in the adventitia of pulmonary arteries (PAs) have been observed in humans and animal models of chronic hypoxic pulmonary hypertension. We have shown that proliferation and apoptosis contribute to this remodeling response. We tested the hypothesis that hypoxia, in the absence of exogenous comitogens, acts directly on PA adventitial fibroblasts to induce proliferation and apoptosis and that the responses are dependent on differential activation of mitogen-activated protein (MAP) kinase family members.

Influence of age on L-type Ca 2+ channels in the pulmonary artery and vein of spontaneously hypertensive rats

The influence of age on the density and localization of L-type Ca 2+ channels was studied during development of hypertension in the pulmonary artery and vein of spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar–Kyoto (WKY) rats by radioligand binding assay and light microscope autoradiography. SHR were examined at 6 weeks (juvenile, pre-hypertensive stage), 12 weeks (young, developing hypertension) and 24 weeks (mature, established hypertension). The dihydropyridine-type Ca 2+ antagonist [ 3H]nicardipine was used as a radioligand. It was bound specifically to sections of rat pulmonary artery and vein. Dissociation constant ( K d) values were similar in WKY rats and SHR, whereas maximum density of binding sites ( B max) values increased in SHR in comparison with WKY rats. This increase was noticeable from the pre-hypertensive phase. The pharmacological profile of [ 3H]nicardipine binding was similar in different age groups of either normotensive and hypertensive rats. Quantitative analysis of autoradiographs from SHR revealed a progressive increase of silver grains in smooth muscle of tunica media and to a lesser extent in the adventitia of pulmonary artery but not of pulmonary vein from pre-hypertensive stage to developing hypertension. No further changes were observed in established hypertension. The above data indicate that the density of L-type Ca 2+ channels of pulmonary arteries is increased in SHR. This augmentation after the pre-hypertensive phase suggests the occurrence of dysregulation of Ca 2+ handling in the pulmonary vasculature of developing SHR.

Immunohistochemical localization of type I, II, III, and IV collagens in the lung.

Type specific rabbit antibodies to bovine type I, II, III, and IV (basement membrane) collagens showing no cross-reaction with other types of collagen were prepared by cross-adsorption and diethylaminoethyl-cellulose chromatography. The antibodies to bovine type I and III collagens showed a high cross-reaction with the corresponding human collagens, but those to type II and IV collagens did moderate and no cross-reactions with human type II and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. both type I and III collagens were found to distribute widely in the interstitium of bronchial tree, bronchial lamina propria and of interlobules as well as alveolar nipples and adventitia of pulmonary arteries. Type II collagen was located only in bronchial cartilage. The tissues mainly stained for type III collagen were the alveolar interstitium (also stained faintly for type I collagen) and the intima and media of the arteries. Type IV collagen was located in a membranous fashion in alveolar septa and bronchial smooth muscles and subepithelial layers as well as capillaries and the intima and media of arteries.