Effects of hypoxia and hyperoxia on the differential expression of VEGF-A isoforms and receptors in Idiopathic Pulmonary Fibrosis (IPF) (original) (raw)
Related papers
American Journal of Respiratory and Critical Care Medicine, 2007
Rationale: Despite intense research efforts, the etiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Objectives: To discover novel genes and/or cellular pathways involved in the pathogenesis of the disease. Methods: We performed expression profiling of disease progression in a well-characterized animal model of the disease. Differentially expressed genes that were identified were compared with all publicly available expression profiles both from human patients and animal models. The role of hypoxia-inducible factor (HIF)-1a in disease pathogenesis was examined with a series of immunostainings, both in the animal model as well as in tissue microarrays containing tissue samples of human patients, followed by computerized image analysis. Measurements and Main Results: Comparative expression profiling produced a prioritized gene list of high statistical significance, which consisted of the most likely disease modifiers identified so far in pulmonary fibrosis. Extending beyond target identification, a series of meta-analyses produced a number of biological hypotheses on disease pathogenesis. Among them, the role of HIF-1 signaling was further explored to reveal HIF-1a overexpression in the hyperplastic epithelium of fibrotic lungs, colocalized with its target genes p53 and Vegf. Conclusions: Comparative expression profiling was shown to be a highly efficient method in identifying deregulated genes and pathways. Moreover, tissue microarrays and computerized image analysis allowed for the high-throughput and unbiased assessment of histopathologic sections, adding substantial confidence in pathologic evaluations. More importantly, our results suggest an early primary role of HIF-1 in alveolar epithelial cell homeostasis and disease pathogenesis, provide insights on the pathophysiologic differences of different interstitial pneumonias, and indicate the importance of assessing the efficacy of pharmacologic inhibitors of HIF-1 activity in the treatment of pulmonary fibrosis.
Respiratory Research
Background: Idiopathic pulmonary fibrosis (IPF) is an age-related, progressive and lethal disease, whose pathogenesis is associated with fibroblasts/myofibroblasts foci that produce excessive extracellular matrix accumulation in lung parenchyma. Hypoxia has been described as a determinant factor in its development and progression. However, the role of distinct members of this pathway is not completely described. Methods: By western blot, quantitative PCR, Immunohistochemistry and Immunocitochemistry were evaluated, the expression HIF alpha subunit isoforms 1, 2 & 3 as well, as their role in myofibroblast differentiation in lung tissue and fibroblast cell lines derived from IPF patients. Results: Hypoxia signaling pathway was found very active in lungs and fibroblasts from IPF patients, as demonstrated by the abundance of alpha subunits 1 and 2, which further correlated with the increased expression of myofibroblast marker αSMA. In contrast, HIF-3α showed reduced expression associated with its promoter hypermethylation. Conclusions: This study lends further support to the involvement of hypoxia in the pathogenesis of IPF, and poses HIF-3α expression as a potential negative regulator of these phenomena.
Hypoxia Inducible Factor 1A Supports a Pro-Fibrotic Phenotype Loop in Idiopathic Pulmonary Fibrosis
International Journal of Molecular Sciences
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The IPF-conditioned matrix (IPF-CM) system enables the study of matrix–fibroblast interplay. While effective at slowing fibrosis, nintedanib has limitations and the mechanism is not fully elucidated. In the current work, we explored the underlying signaling pathways and characterized nintedanib involvement in the IPF-CM fibrotic process. Results were validated using IPF patient samples and bleomycin-treated animals with/without oral and inhaled nintedanib. IPF-derived primary human lung fibroblasts (HLFs) were cultured on Matrigel and then cleared using NH4OH, creating the IPF-CM. Normal HLF-CM served as control. RNA-sequencing, PCR and western-blots were performed. HIF1α targets were evaluated by immunohistochemistry in bleomycin-treated rats with/without nintedanib and in patient samples with IPF. HLFs cultured on IPF-CM showed over-expression of ‘HIF1α signaling pathway’ (KEGG, p < 0.0001), ...
Evaluation of lung VEGF-A transduction during hyperoxia-induced injury in rats
2018
BackgroundSince Vascular Endothelial Growth Factor (VEGF) is a main factor for endothelial survival, we evaluated whether VEGF transduction could ameliorate hyperoxia induced injury, which is associated with predominant endothelial injury.Methods and ResultsTransduction (induced 48 hours before hyperoxic exposure) using adenoviral vector (Ad.) for VEGF (1010 viral particles [VP]) increased moderately survival under hyperoxia (fraction of inspired oxygen [FIO2] >95%) as compared with Ad.Null (1010 VP) transduction, whereas VEGF transduction with a lower dose (5.109 VP) had no effect. After 48 hours of hyperoxia, Ad.VEGF transduction increased lung VEGF concentration, prevented the diffuse loss of capillary bed and induced patchy areas of endothelial cell proliferation (CD31 immunostaining) with interstitial inflammatory cell recruitment as compared to Ad. Null transduction. Hyperoxia was associated with diffuse apoptosis that was inhibited only in patchy areas of endothelial proli...
Hypoxia upregulates VEGF expression in alveolar epithelial cells in vitro and in vivo
American Journal of Physiology - Lung Cellular and Molecular Physiology, 2002
We investigated regulation of vascular endothelial growth factor (VEGF) expression by hypoxia in cultured and freshly isolated rat alveolar epithelial cells (AEC). In vitro, hypoxia increased VEGF mRNA and protein levels, with maximal stimulation at 0% O2 for 18 h. A similar upregulation of VEGF expression was found in alveolar epithelial type II (ATII) cells freshly isolated from rats exposed to 8% O2 for 24 h. In vitro, hypoxia-induced upregulation of VEGF mRNA was due to an increase in transcription, rather than an increase in RNA stability, inasmuch as the half-life of VEGF mRNA was unchanged. Upregulation of VEGF mRNA by hypoxia was mimicked by CoCl2 and desferrioxamine in normoxic AEC and was not prevented by inhibitors of reactive oxygen species, suggesting that hypoxic VEGF regulation involved an O2-dependent protein that requires ferrous ions but is independent of reactive oxygen species generation. In polarized ATII cells, VEGF protein was secreted at the apical and basolateral sides. Similarly, in rats, VEGF was secreted in bronchoalveolar lavage fluid. Hypoxia induced a twofold increase in VEGF protein at the apical side of ATII cells in culture and in bronchoalveolar lavage fluid. These findings suggest that release of VEGF synthesized by AEC may target not only endothelial cells but also other alveolar cells, including macrophages and epithelial cells.
Cells
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control′s response is greater by the number of genes and complexity. In the search fo...
Cytokine, 2003
Chronic hypoxia is implicated in lung fibrosis, which is characterized by enhanced deposition of extracellular matrix (ECM) molecules. Transforming growth factor-b (TGF-b) plays a key role in fibroblast homeostasis and is involved in disease states characterized by excessive fibrosis, such as pulmonary fibrosis. In this study, we investigated if hypoxia modulates the effects of TGF-b on the expression of gelatinases: matrix metalloproteinase (MMP)-2 and MMP-9, interstitial collagenases: MMP-1 and MMP-13, tissue inhibitors of MMP (TIMP), collagen type I and interleukin-6 (IL-6). Primary human lung fibroblasts, established from tissue biopsies, were cultivated under normoxia or hypoxia in the presence of TGF-b1, TGF-b2 or TGF-b3. Gelatinases were assessed by gelatin zymography and collagenases, TIMP, collagen type I and IL-6 by ELISA. Under normoxia fibroblasts secreted MMP-2, collagenases, TIMP, collagen type I and IL-6. TGF-bs significantly decreased MMP-1 and increased TIMP-1, IL-6 and collagen type I. Hypoxia significantly enhanced MMP-2, and collagenases. Compared to normoxia, the combination of TGF-b and hypoxia reduced MMP-1, and further amplified the level of TIMP, IL-6, and collagen type I. Thus, in human lung fibroblasts hypoxia significantly increases the TGF-bs-induced secretion of collagen type I and may be associated to the accumulation of ECM observed in lung fibrosis.
The FASEB Journal, 2005
Proliferation of adventitial fibroblasts of small intrapulmonary arteries (FB PA) 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 FB PA exposed to hypoxia. Primary cultures of FB PA displayed a strong mitogenic response to 24 h hypoxia, whereas the rate of apoptosis was significantly suppressed. In addition, the migration of FB PA was strongly increased under hypoxic conditions but not the expression of α-smooth muscle actin. Hypoxia induced a marked up-regulation (protein level) of both HIF-1α and HIF-2α, alongside with nuclear translocation of these transcription factors. Specific inhibition of either HIF-1α or HIF-2α was achieved by RNA interference technology, as proven by HIF-1α and HIF-2α 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 FB PA was found to be solely HIF-2α dependent, whereas the migratory response was significantly reduced by both HIF-1α and HIF-2α 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-2α playing a predominant role, which may offer future intervention strategies. Key words: adventitia • α-smooth-muscle actin • remodeling • RNA interference rolonged alveolar hypoxia, as occurs, e.g., at high altitude, in chronic obstructive pulmonary disease and in various restrictive lung diseases, results in a remodeling of the pulmonary vasculature, which is characterized by proliferative changes in the intima, media, and adventitia of the pulmonary artery. These changes may cause chronic pulmonary arterial hypertension and subsequent cor pulmonale. P
Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling
Scientific reports, 2018
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduc...