Karl Sanders

NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-β1 and insulin-like growth factor binding protein-3

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

Receptors for Advanced Glycation End-Products Targeting Protect against Hyperoxia-Induced Lung Injury in Mice

Patients with acute lung injury almost always require supplemental oxygen during treatment; however, elevated oxygen itself is toxic. Receptors for advanced glycation end-products (RAGE) are multi-ligand cell surface receptors predominantly localized to alveolar type I cells that influence development and cigarette smoke-induced inflammation, but studies that address the role of RAGE in acute lung injury are insufficient. In the present investigation, we test the hypothesis that RAGE signaling functions in hyperoxia-induced inflammation. RAGE-null mice exposed to hyperoxia survived 3 days longer than age-matched wild-type mice. After 4 days in hyperoxia, RAGE-null mice had less total cell infiltration into the airway, decreased total protein leak, diminished alveolar damage in hematoxylin and eosin-stained lung sections, and a lower lung wet-to-dry weight ratio. An inflammatory cytokine antibody array revealed decreased secretion of several proinflammatory molecules in lavage fluid obtained from RAGE knockout mice when compared with wild-type control animals. Real-time RT-PCR and immunoblotting revealed that hyperoxia induced RAGE expression in primary alveolar epithelial cells, and immunohistochemistry identified increased RAGE expression in the lungs of mice after exposure to hyperoxia. These data reveal that RAGE targeting leads to a diminished hyperoxia-induced pulmonary inflammatory response. Further research into the role of RAGE signaling in the lung should identify novel targets likely to be important in the therapeutic alleviation of lung injury and associated persistent inflammation.

The Role of Endogenous NADPH Oxidases in Airway and Pulmonary Vascular Smooth Muscle Function

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O(2) sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.

The role of NADPH oxidase in carotid body arterial chemoreceptors.

O(2)-sensing in the carotid body occurs in neuroectoderm-derived type I glomus cells where hypoxia elicits a complex chemotransduction cascade involving membrane depolarization, Ca(2+) entry and the release of excitatory neurotransmitters. Efforts to understand the exquisite O(2)-sensitivity of these cells currently focus on the coupling between local P(O2) and the open-closed state of K(+)-channels. Amongst multiple competing hypotheses is the notion that K(+)-channel activity is mediated by a phagocytic-like multisubunit enzyme, NADPH oxidase, which produces reactive oxygen species (ROS) in proportion to the prevailing P(O2). In O(2)-sensitive cells of lung neuroepithelial bodies (NEB), multiple studies confirm that ROS levels decrease in hypoxia, and that E(M) and K(+)-channel activity are indeed controlled by ROS produced by NADPH oxidase. However, recent studies in our laboratories suggest that ROS generated by a non-phagocyte isoform of the oxidase are important contributors to chemotransduction, but that their role in type I cells differs fundamentally from the mechanism utilized by NEB chemoreceptors. Data indicate that in response to hypoxia, NADPH oxidase activity is increased in type I cells, and further, that increased ROS levels generated in response to low-O(2) facilitate cell repolarization via specific subsets of K(+)-channels.

The NOX on Pulmonary Hypertension

See related article, pages 258–267 Chronic pulmonary arterial hypertension (PAH) is a devastating clinical disorder that contributes to the morbidity and mortality of adult and pediatric patients with a wide range of lung and heart diseases. Diseases leading to pulmonary hypertension are frequently associated with hypoxia within discrete areas of the lung. Acutely, the regional response to hypoxia is a reversible contraction of pulmonary artery smooth muscle cells (PASMC), which is a protective physiologic response that serves to redirect blood to better-ventilated areas of the lung. This constrictor response of PASMC contrasts with that of systemic arterial smooth muscle cells, which usually relax in response to hypoxia, indicating that oxygen (O2) sensing mechanisms in vascular smooth muscle are adapted to the environment from which they are derived. Importantly, chronic hypoxia induces irreversible changes of profound vascular remodeling characterized by medial and adventitial thickening of the muscular and elastic vessels and muscularization of previously nonmuscularized more distal small vessels. This is the basis for debilitating persistent PAH.1 Reactive oxygen species (ROS) are important regulators of vascular tone and function.2,3 In the lung, ROS are implicated in acute hypoxic vasoconstriction.4 Administration of superoxide dismutase significantly attenuates pulmonary vasoconstriction because of hypoxia.5 Moreover, several studies have now shown that agents promoting ROS generation stimulate both systemic arterial smooth muscle cells and PASMC proliferation implicating ROS in the vascular remodeling associated with chronic hypoxia. Again, suppression of endogenous ROS production inhibits smooth muscle cell (SMC) proliferation and promotes apoptosis.6–8 In animal models, ROS production has been directly linked to the vascular remodeling associated …

Carotid body chemoreceptor activity in mice deficient in selected subunits of NADPH oxidase.

Exposure of the carotid body to hypoxia elicits increased neural activity in the carotid sinus nerve (CSN), and reflex cardio-pulmonary adjustments which mitigate the adverse effects of hypoxemia. Increased carotid body activity occurs at relatively moderate arterial P02, in contrast to the severe hypoxia required to elicit metabolic and functional adjustments in non-02 sensing tissues (S.J.Fidone et al. 1997). Chemosensory type I cells derived from neuroectoderm are responsible for this exquisite sensitivity, and numerous laboratories have reported that low P02 inhibits the conductance of a variety of voltage sensitive and voltage-insensitive K+-channels in these cells. Yet the molecular mechanism underlying the P02 modulation of cell currents remains uncertain and controversial (H.Acker et al 1994, A.M.Riesco-Fagundo et al2001). Various heme proteins have been proposed as primary O2 sensors, and one set of data in particular suggests the involvement of a multi-component cytochrome b-containing NADPH oxidase which may be similar if not identical to the superoxide generating enzyme commonly found in phagocytic cells (H.Acker et al. 1994) (H.Acker et al. 1994), but the relationship between PO2 and ROS levels in type I cells has not been firmly established. In other cells and tissues hypoxia can increase or decrease ROS production in either mitochondria or via NADPH oxidase (I.O’Kelly et al. 2000, G.B Waypa et al. 2001). In addition, the target of ROS in type I cells is an unknown and critical factor in determining the effect of NADPH oxidase on cell activity. Recent studies have indicated that voltage-sensitive K+-channels in type I cells are modulated by hypoxia via a mechanism independent of soluble factors such as ROS (A.M. Riesco-Fagundo et al. 2001). Thus ROS do not appear to be necessary for cell activation. On the other hand, if hypoxia enhances NADPH oxidase activity, elevated ROS levels may increase the open probability of K+-channels thus facilitating cell repolarization. Such a scheme is consistent with elevated CSN activity in p47phox-gene deleted animals. Clarification of these issues must await future measurements of the effect of hypoxia on NADPH oxidase activity, and evaluation of the interaction of ROS with the chemotransduction machinery in type I cells.