Irradiation-dependent effects on tumor perfusion and endogenous and exogenous hypoxia markers in an A549 xenograft model.

PURPOSE: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. MATERIALS AND METHODS: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. RESULTS: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. CONCLUSIONS: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

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.

Analysis of tumor vessel supply in Lewis lung carcinoma in mice by fluorescent microsphere distribution and imaging with micro- and flat-panel computed tomography.

In lung carcinomas the blood supply varies depending on tumor type and stage and can develop from pulmonary or bronchial circulation, or both. To examine this in vivo, primary bronchogenic Lewis lung carcinoma cells were intratracheally instilled in C57BL/6 mice. Within 7 days, histological examinations showed progressive tumor growth at the peripheral parenchymal region. The relative contribution of tumor blood supply via the pulmonary and systemic arteries was studied in detail using fluorescent microspheres (10 microm). When compared to healthy lung parenchyma (13:1), Lewis lung carcinoma tumor tissue (52:1) showed a fourfold increase in pulmonary to systemic microspheres, indicating that the pulmonary arteries are the predominant tumor-feeding vessels. After filling the vessels with a vascular cast, the microanatomy of vessels being derived from the pulmonary artery was visualized with micro computed tomography. Flat-panel volumetric computed tomography provided longitudinal visualization of tissue bridges between the growing tumor and the pulmonary vasculature. In this model of peripheral parenchymal malignancy, new imaging techniques allowed effective visualization of lung tumor growth and vascularization in living mice, demonstrating a pulmonary blood supply for lung tumors.

Role of hypoxia-inducible factor-1alpha in hypoxia-induced apoptosis of primary alveolar epithelial type II cells.

Hypoxia affects alveolar homeostasis and may induce epithelial injury, which has been implicated in lung diseases such as fibrosis. The underlying cellular and molecular mechanisms are, however, largely unknown. Primary rat alveolar epithelial type II cells (ATII) exposed to graded hypoxia for 24 and 48 h caused a dose-dependent induction of cell cycle arrest and suppression of proliferation, which were comparable to the effects of angiotensin II, a potent inducer of ATII cell death. Hypoxia-induced changes in ATII homeostasis are thought to proceed primarily via activation of hypoxia inducible-factor (HIF)-1alpha, because hypoxia increased HIF-1alpha protein expression, nuclear translocation, and transactivation of its specific DNA binding domain, the hypoxia responsive element (HRE). Under hypoxic conditions, expression of the proapoptotic protein Bnip3L, which belongs to the Bcl 2 family and is known to be one of the HIF-1-dependent target genes, was upregulated. Suppression of HIF-1alpha or Bnip-3L with small interfering RNA (siRNA) fully blocked the hypoxia-induced apoptosis and Bnip3L expression. In line with these data, overexpression of HIF-1alpha by transient transfection enhanced the hypoxia-induced apoptosis. Thus, we conclude that hypoxia suppresses alveolar epithelial cell proliferation and enhances ATII apoptosis through activation of the HIF-1alpha/HRE axis and a mechanism that involves Bnip3L. Targeting HIF-1alpha may represent a new strategy that could impede the alveolar denudation that is observed in several lung diseases.

RNA interference for HIF-1alpha inhibits its downstream signalling and affects cellular proliferation.

Transcription factor HIF-1 is a key determinant of oxygen-dependent gene regulation. Suppression of HIF-1alpha is important for exploring HIF-1-dependent processes and for interfering with hypoxia-induced pathophysiological events. This study applied RNA-interference targeting HIF-1alpha to the human lung A549 cell line. Transfection of HIF-1alpha-siRNA reduced HIF-1alpha synthesis as measured on mRNA and protein level by realtime RT-PCR, Western blot, and immuncytochemistry. A time kinetic for hypoxic stabilization of HIF-1alpha protein and its inhibition by HIF-1alpha-siRNA is included. Hypoxic induction of HIF-1-controlled target genes as heme oxygenase I (HO-1), phosphoglycerate kinase (PGK), and vascular endothelial growth factor (VEGF) was markedly attenuated by HIF-1alpha-siRNA treatment. Correspondingly, gene activation via hypoxia-responsive-element, as shown by reporter gene assay, was inhibited by HIF-1alpha-siRNA. Moreover, this approach was found to suppress the shift from from S-phase to G1-phase observed in A549 cells in response to hypoxia, supporting a role of HIF-1alpha in oxygen-dependent cell cycle regulation.