Pulmonary Artery Adventitial Fibroblasts Research Articles

Fibroblasts in Pulmonary Hypertension: Roles and Molecular Mechanisms.

Fibroblasts, among the most prevalent and widely distributed cell types in the human body, play a crucial role in defining tissue structure. They do this by depositing and remodeling extracellular matrixes and organizing functional tissue networks, which are essential for tissue homeostasis and various human diseases. Pulmonary hypertension (PH) is a devastating syndrome with high mortality, characterized by remodeling of the pulmonary vasculature and significant cellular and structural changes within the intima, media, and adventitia layers. Most research on PH has focused on alterations in the intima (endothelial cells) and media (smooth muscle cells). However, research over the past decade has provided strong evidence of the critical role played by pulmonary artery adventitial fibroblasts in PH. These fibroblasts exhibit the earliest, most dramatic, and most sustained proliferative, apoptosis-resistant, and inflammatory responses to vascular stress. This review examines the aberrant phenotypes of PH fibroblasts and their role in the pathogenesis of PH, discusses potential molecular signaling pathways underlying these activated phenotypes, and highlights areas of research that merit further study to identify promising targets for the prevention and treatment of PH.

Microenvironmental Regulation of Macrophage Transcriptomic and Metabolomic Profiles in Pulmonary Hypertension.

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro, and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression.

Pirfenidone Inhibits Hypoxic Pulmonary Hypertension through the NADPH/ROS/p38 Pathway in Adventitial Fibroblasts in the Pulmonary Artery.

Hypoxic pulmonary hypertension (HPH) is a devastating disease characterized by progressive vasoconstriction and vascular remodeling. Pirfenidone (PFD) inhibits the progression of HPH, though the molecular mechanisms remain unknown. This study is aimed at determining the role and mechanism of PFD in HPH in human pulmonary artery adventitial fibroblasts (HPAAFs), which were cultured under normal or hypoxic conditions. NOX4 and Rac1 were inhibited or overexpressed by shRNA or pcDNA3.1, respectively. Proliferation of HPAAFs was quantified by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assays to assess cellular metabolic activity, cell counts, and ethynyldeoxyuridine (EdU) assays to detect DNA synthesis. Migration of HPAAFs was assessed by a wound healing assay. The expression levels of smooth muscle alpha-actin (a-SMA) and procollagen I (COL1A1) were assessed by RT-PCR and western blot analysis. PFD suppressed hypoxia-induced proliferation and migration of HPAAFs. Compared with the hypoxic control group, PFD reduced the expression of a-SMA and procollagen I (COL1A1). PFD reduced hypoxia-induced phosphorylation of p38 through the NOX4/reactive oxygen species (ROS) signaling pathway. Moreover, Rac1 also decreased hypoxia-induced phosphorylation of p38, without any cross-interaction with NOX4. These findings demonstrate that PFD is a novel therapeutic agent to prevent cell proliferation, migration, and fibrosis, which might be useful in inhibiting vascular remodeling in patients with HPH.

RNA-binding protein SFPQ cooperates with HDAC1 to suppress CD40 transcription in pulmonary adventitial fibroblasts.

Pulmonary artery adventitial fibroblasts, the most abundant cellular constituent of adventitia, are often the first to be activated and reprogrammed to then influence the tone and structure of the vessel wall in pulmonary arterial hypertension (PAH). Our previous study found that interruption of CD40 ligand (CD40L)-CD40 signaling improves the efficacy of transplanted endothelial progenitor cells in monocrotaline induced-PAH. However, whether CD40L-CD40 signaling is involved in the activation of adventitial fibroblasts in PAH and whether Drosophila behavior human splicing (DBHS) protein family members have any roles during adventitial fibroblasts activation are completely unclear. Here, we show that soluble CD40L (sCD40L) stimulation progressively increases pro-inflammatory activity, proliferation, and migration of pulmonary adventitial fibroblasts. Besides, sCD40L stimulation decreases splicing factor proline- and glutamine-rich protein (SFPQ) protein (one member of DBHS protein family) expression, while SFPQ overexpression suppresses sCD40L stimulation-induced proliferation and migration of pulmonary adventitial fibroblasts by repressing CD40 transcription. Moreover, ChIP assays found that sCD40L stimulation promotes histone H3 tri-methylation at lysine 4 (H3K4me3), H3K36me3, and H3K27 acetylation (H3K27ac) modifications on CD40 promoter region in pulmonary adventitial fibroblasts, while SFPQ overexpression decreases H3K36me3 modification and increases H3K36ac on CD40 promoter region by interacting with histone deacetylase-1 (HDAC1) to inhibit CD40 transcription. This in-depth study shows that CD40L-CD40 signaling promotes activation of pulmonary adventitial fibroblasts by increasing proliferation, migration, and pro-inflammatory activity of adventitial fibroblasts, and SFPQ could inhibit CD40 transcription though switching H3K36me3 to H3K36ac modifications on its promoter by interacting with HDAC1. This study, first, uncovers the roles of SFPQ on CD40L-CD40 signaling-mediated activation of pulmonary adventitial fibroblasts.

Transglutaminase 2 in pulmonary and cardiac tissue remodeling in experimental pulmonary hypertension.

Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.

Serotonin drives the activation of pulmonary artery adventitial fibroblasts and TGF-β1/Smad3-mediated fibrotic responses through 5-HT(2A) receptors.

Pulmonary arterial remodeling is characterized by excessive proliferation, migration, and pro-differentiation and fibrotic activation of adventitial fibroblasts in pulmonary arterial hypertension (PAH) process. Several lines of evidence indicate that serotonin (5-HT) plays a central role in the pathogenesis of pulmonary arterial remodeling. In the present study, we investigated whether 5-HT is directly involved in the functional regulation of pulmonary artery adventitial fibroblasts (PAFs). Incubation of cultured rat PAFs with 5-HT caused a dose-dependent stimulation of cell proliferation, migration activity, and a time-dependent increase of α-SMA expression, a marker of fibroblast differentiation into myofibroblasts, and adventitia fibrosis, evaluating connective tissue growth factor (CTGF) and extracellular matrix (ECM) mRNAs and proteins. These effects were attenuated by the 5-HT2A receptor antagonist, ketanserin and mimicked by the 5-HT2A receptor agonist DOI. 5-HT-induced fibroblasts phenotypic alterations and ECM accumulation were dependent on stimulation of transforming growth factor (TGF)-β1 as demonstrated using a neutralizing antibody. 5-HT also caused Smad3 phosphorylation and ketanserin diminished 5-HT-induced Smad3 activation. These results demonstrated that 5-HT can directly activate PAFs through 5-HT2A receptor and promote fibroblasts phenotypic alterations and adventitia fibrosis depending on the signaling of the TGF-β1/Smad3 pathway.

Pulmonary artery adventitial fibroblasts acquire JNK1‐mediated proliferative responses upon exposure to chronic hypoxia

Pulmonary artery adventitial fibroblasts acquire JNK1‐mediated proliferative responses upon exposure to chronic hypoxia

Superoxide enhances cell proliferation in pulmonary artery adventitial fibroblasts from chronically hypoxic calves and augments expression of the redox‐sensitive transcription factor, Egr‐1

Superoxide (O2−) has been implicated in chronic hypoxia‐induced pulmonary hypertension (CHPH) and pulmonary vascular remodeling. In a calf model of CHPH, the pulmonary artery adventitial fibroblast (PAAF) proliferates in vivo and in vitro and contributes to vascular remodeling. Hypoxia‐induced proliferation of PAAF is mediated in part by a redox‐sensitive transcription factor, early growth response‐1 (Egr‐1). We hypothesized that ROS are responsible for the proliferation observed in PAAF from the chronically hypoxic (HA) calf through Egr‐1. We cultured PAAF isolated from HA neonatal calves in the presence or absence of the antioxidant, superoxide dismutase (SOD, 200 U/ml) for up to 6 days and measured changes in cell counts by hemocytometer and MTT assay. We exposed cells to xanthine and xanthine oxidase (8 mU/mL) to generate O2−, and measured Egr‐1 mRNA expression by real‐time PCR. We found that SOD decreased proliferation in PAAF from HA calves. Treatment of HA PAAF with xanthine/xanthine oxidase increased Egr‐1 mRNA expression by 2 fold (p<0.05 by unpaired t‐test). These data provide evidence that O2− contributes to the proliferation of PAAF from HA calves and support the hypothesis that O2− may regulate cell proliferation by upregulating the transcription factor, Egr‐1.

NOX4 Regulates ROS Levels Under Normoxic and Hypoxic Conditions, Triggers Proliferation, and Inhibits Apoptosis in Pulmonary Artery Adventitial Fibroblasts

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

Hypoxia exposure induces the emergence of fibroblasts lacking replication repressor signals of PKC in the pulmonary artery adventitia

Cultured fibroblasts of hypoxia-stimulated remodelled pulmonary artery (PA) adventitia proliferate at a greater rate compared with those of normal adventitia. Since protein kinase C (PKC) zeta is a replication repressor of normal adventitial fibroblasts, we hypothesized that loss of the repressor activity of PKCzeta might contribute to increased rate of proliferation in adventitial cells of remodelled PA. Isolated PA adventitial fibroblasts of neonatal control (Fib-C) and chronic hypoxia-exposed (Fib-H) calves were used to test our hypothesis. For evaluation of the role of PKCzeta in hypoxia-induced vascular adventitial remodelling, expression and activation of PKCzeta were also examined in lung sections of Fib-C and Fib-H animals by immunoperoxidase staining. Although constitutively active PKCzeta expression attenuated DNA synthesis in Fib-C, it stimulated proliferation in Fib-H. PKCzeta-specific myristoylated pseudosubstrate peptide inhibitor (PKCzeta-PI) induced replication in Fib-C, whereas the inhibitor blocked DNA synthesis in Fib-H. Hypoxia stimulated PKCzeta as well as MAP kinase kinase (MEK)1/2 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation in Fib-H cells. However, ERK1/2 activation was mediated by both MEK1/2-dependent and MEK1/2-independent PKCzeta-regulated mechanisms in hypoxia-exposed Fib-H. PKCzeta was selectively activated in the adventitial cells of the remodelled vascular wall, as demonstrated by strong immunoreactivity against the anti-phosphoPKCzeta antibody in the Fib-H lung sections. PKCzeta acts as a replication repressor in Fib-C cells; however, the same isozyme mediates Fib-H proliferation. Thus, chronic exposure to hypoxia leads to the emergence of cells lacking anti-replication activity of PKCzeta in the PA adventitia.

P38 MAP kinase: Essential role in hypoxia-mediated human pulmonary artery fibroblast proliferation

Pulmonary arterial hypertension (PAH) is a disease that results in thickening of the vascular wall. Some of the most prominent changes are seen in the adventitia as a result of fibroblast proliferation and increased extracellular matrix deposition. Previous work from this laboratory using animal models has shown that pulmonary but not systemic artery fibroblasts proliferate to hypoxic exposure and that this response is dependent on activation of p38 mitogen-activated protein kinase (p38MAPK). In this study, we wished to determine whether human pulmonary artery fibroblasts (HPAFs) behaved similarly under conditions of acute hypoxic exposure (35 mmHg for 24 h). Fibroblast proliferation was assessed by [ 3H]thymidine uptake and protein assays performed using Western blotting techniques. HPAFs proliferated in response to acute hypoxic exposure, human systemic artery fibroblasts did not. This hypoxia-mediated proliferation was p38 MAPK dependent and could be blocked using a specific p38 MAPK inhibitor. Hypoxia-inducible factor-1 (HIF-1) expression was increased in hypoxic pulmonary but not systemic cells and could be partially abrogated with the p38 inhibitor. This work in man confirmed our previous findings in animals that significant differences exist between the pulmonary and systemic circulations in response to hypoxic exposure. This study highlights the importance of p38 MAPK and HIF-1 in hypoxia-mediated proliferation of pulmonary artery adventitial fibroblasts.

Pulmonary Artery Adventitial Fibroblasts Cooperate with Vasa Vasorum Endothelial Cells to Regulate Vasa Vasorum Neovascularization: A Process Mediated by Hypoxia and Endothelin-1

The precise cellular and molecular mechanisms regulating adventitial vasa vasorum neovascularization, which occurs in the pulmonary arterial circulation in response to hypoxia, remain unknown. Here, using a technique to isolate and culture adventitial fibroblasts (AdvFBs) and vasa vasorum endothelial cells (VVECs) from the adventitia of pulmonary arteries, we report that hypoxia-activated pulmonary artery AdvFBs exhibited pro-angiogenic properties and influenced the angiogenic phenotype of VVEC, in a process of cell-cell communication involving endothelin-1 (ET-1). We demonstrated that AdvFBs, either via co-culture or conditioned media, stimulated VVEC proliferation and augmented the self-assembly and integrity of cord-like networks that formed when VVECs where cultured on Matrigel. In addition, hypoxia-activated AdvFBs produced ET-1, suggesting a paracrine role for this pro-angiogenic molecule in these processes. When co-cultured on Matrigel, AdvFBs and VVECs self-assembled into heterotypic cord-like networks, a process augmented by hypoxia but attenuated by either selective endothelin receptor antagonists or oligonucleotides targeting prepro-ET-1 mRNA. From these observations, we propose that hypoxia-activated AdvFBs exhibit pro-angiogenic properties and, as such, communicate with VVECs, in a process involving ET-1, to regulate vasa vasorum neovascularization occurring in the adventitia of pulmonary arteries in response to chronic hypoxia.

Impact of HIF‐1α and HIF‐2α on proliferation and migration of human pulmonary artery fibroblasts in hypoxia

Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FBPA) has been disclosed as an early event in the development of pulmonary hypertension and cor pulmonale in response to hypoxia. We investigated the role of hypoxia-inducible transcription factors (HIF) in human FBPA exposed to hypoxia. Primary cultures of FBPA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FBPA was strongly increased under hypoxic conditions but not the expression of alpha-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1alpha and HIF-2alpha, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1alpha or HIF-2alpha was achieved by RNA interference technology, as proven by HIF-1alpha and HIF-2alpha mRNA and protein analysis and expression analysis of HIF downstream target genes. With the use of this approach, the hypoxia-induced proliferative response of the FBPA was found to be solely HIF-2alpha dependent, whereas the migratory response was significantly reduced by both HIF-1alpha and HIF-2alpha interference. In conclusion, HIF up-regulation is essential for hypoxic cellular responses in human pulmonary artery adventitial fibroblasts such as proliferation and migration, mimicking the pulmonary hypertensive phenotype in vivo. Differential HIF subtype dependency was noted, with HIF-2alpha playing a predominant role, which may offer future intervention strategies.

Hypoxia‐driven proliferation of human pulmonary artery fibroblasts: cross‐talk between HIF‐1α and an autocrine angiotensin system

Pulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and cor pulmonale. In cultured human FBPA, both angiotensin II (Ang II) and hypoxia promoted cell cycle progression and cell proliferation and suppressed apoptosis. These effects were further enhanced when both stimuli were applied simultaneously. Hypoxia elevated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased the expression of genes regulated by the hypoxia-responsive element (HRE). Up-regulation of both angiotensin-converting enzyme (ACE) and Ang II receptor type 1 (AT1) was also observed. Exogenous Ang II further increased HIF/HRE-dependent signaling in FBPA, whereas suppression of the autocrine ACE-Ang II-AT1 loop with inhibitors of ACE, AT1, and phosphatidylinositol 3-kinase (PI3K) reduced the proliferative response to both hypoxia and exogenous Ang II. Overexpression of HIF-1alpha by transient transfection caused the same proliferative effect and up-regulation of AT1 expression that were observed under hypoxic conditions. In contrast, small interfering RNA targeting HIF-1alpha inhibited hypoxia-induced ACE and AT1 expression. Our studies indicate that the ACE-Ang II-AT1 system serves as a positive feedback loop and fosters FBPA proliferation under hypoxic conditions, with the PI3K-HIF-HRE axis as the central effector pathway. This pathway may thus facilitate vascular remodeling under hypoxic conditions.

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

Activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin is necessary for hypoxia-induced pulmonary artery adventitial fibroblast proliferation

In contrast to cell types in which exposure to hypoxia causes a general reduction of metabolic activity, a remarkable feature of pulmonary artery adventitial fibroblasts is their ability to proliferate in response to hypoxia. Previous studies have suggested that ERK1/2, phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) are activated by hypoxia and play a role in a variety of cell responses. However, the pathways involved in mediating hypoxia-induced proliferation are largely unknown. Using pharmacological inhibitors, we established that PI3K-Akt, mTOR-p70 ribosomal protein S6 kinase (p70S6K), and EKR1/2 signaling pathways play a critical role in hypoxia-induced adventitial fibroblast proliferation. We found that exposure of serum-starved fibroblasts to 3% O2 resulted in a time-dependent activation of PI3K and transient phosphorylation of Akt. However, activation of PI3K was not required for activation of ERK1/2, implying a parallel involvement of these pathways in the proliferative response of fibroblasts to hypoxia. We found that hypoxia induced significant increases in mTOR, p70S6K, 4E-BP1, and S6 ribosomal protein phosphorylation, as well as dramatic increases in p70S6K activity. The activation of p70S6K/S6 pathway was sensitive to inhibition by rapamycin and LY294002, indicating that mTOR and PI3K/Akt are upstream signaling regulators. However, the magnitude of hypoxia-induced p70S6K activity and phosphorylation suggests involvement of additional signaling pathways. Thus our data demonstrate that hypoxia-induced adventitial fibroblast proliferation requires activation and interaction of PI3K, Akt, mTOR, p70S6K, and ERK1/2 and provide evidence for hypoxic regulation of protein translational pathways in cells exhibiting the capability to proliferate under hypoxic conditions.

Egr-1 antisense oligonucleotides inhibit hypoxia-induced proliferation of pulmonary artery adventitial fibroblasts.

In most mammalian species, chronic exposure to hypoxia leads to pulmonary hypertension and vascular remodeling. The adventitial fibroblast, because of its ability to proliferate in response to hypoxia, is thought to be a critical cell in the remodeling process. However, the transcription factors driving hypoxia-induced fibroblast proliferation have yet to be elucidated. The early growth response-1 (Egr-1) transcription factor has been shown to be upregulated by hypoxia in pulmonary artery adventitial fibroblasts. We therefore hypothesized that Egr-1 is directly involved in hypoxia-induced adventitial fibroblast proliferation. Immunohistochemical analysis of in vivo lung tissue from animals exposed to chronic hypoxia revealed increased expression of Egr-1 in the pulmonary artery fibroblasts vs. expression shown in normoxic controls. In fibroblasts cultured from chronically hypoxic animals, exposure to 1% oxygen upregulated Egr-1 protein and cell proliferation. To evaluate the role of Egr-1 in hypoxia-induced proliferation, we employed an Egr-1 antisense strategy. Addition of antisense Egr-1 oligonucleotides, but not sense oligonucleotides, attenuated the hypoxia-induced upregulation of Egr-1 protein and reduced hypoxia-induced DNA synthesis by 50%. Cell proliferation was also significantly inhibited by the addition of antisense Egr-1 oligonucleotides but not the sense oligonucleotides. In addition, hypoxia-induced upregulations of cyclin D and epidermal growth factor receptor were attenuated by Egr-1 antisense oligonucleotides. We conclude that Egr-1 protein expression is very sensitive to upregulation by hypoxia in pulmonary artery adventitial fibroblasts and that it plays an important role in the autonomous growth phenotype induced by hypoxia in these cells.

Hypoxia induces differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts.

Activation of the alpha-smooth muscle actin (alpha-SMA) gene during the conversion of fibroblasts into myofibroblasts is an essential feature of various fibrotic conditions. Microvascular compromise and thus local environmental hypoxia are important components of the fibrotic response. The present study was thus undertaken to test the hypothesis that hypoxia can induce transdifferentiation of vascular fibroblasts into myofibroblasts and also to evaluate potential signaling mechanisms governing this process. We found that hypoxia significantly upregulates alpha-SMA protein levels in bovine pulmonary artery adventitial fibroblasts. Increased alpha-SMA expression is controlled at the transcriptional level because the alpha-SMA gene promoter activity, assayed via a luciferase reporter, was markedly increased in transfected fibroblasts exposed to hypoxia. Hypoxic induction of the alpha-SMA gene was mimicked by overexpression of constitutively active Galphai2 (alphai2Q205L) but not Galpha16 (alpha-16Q212L). Blockade of hypoxia-induced alpha-SMA expression with pertussis toxin, a Galphai antagonist, confirmed a role for Galphai in the hypoxia-induced transdifferentiation process. c-Jun NH2-terminal kinase (JNK) inhibitor II and SB202190, but not U0126, also attenuated alpha-SMA expression in hypoxic fibroblasts, suggesting the importance of JNK in the differentiation process. Hypoxia-induced increase in bromodeoxyuridine incorporation, which occurred concomitantly with hypoxia-induced differentiation, was blocked by U0126, suggesting that DNA synthesis and alpha-SMA expression take place through simultaneously activated parallel signaling pathways. Neutralizing antibody against transforming growth factor-beta1 blocked only 30% of the hypoxia-induced alpha-SMA promoter activity. Taken together, our results suggest that hypoxia induces differentiation of vascular fibroblasts into myofibroblasts by upregulating the expression of alpha-SMA, and this increase in alpha-SMA level occurs through Galphai- and JNK-dependent signaling pathways.

Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors.

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co-cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum-free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia-inducible-transcription-factor subtypes (HIF-1alpha, HIF-2alpha, HIF-3alpha) and its dependent target genes, carrying the hypoxia-responsive-element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF-transcription-factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia-driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF-transcription-decoy may offer a new therapeutic approach to suppress these events.

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.