Prolonged hypoxia produces reversible changes in endothelial permeability, but the mechanisms involved are not fully known. Previous studies have implicated reactive oxygen species (ROS) and cytokines in the regulation of permeability. We tested whether prolonged hypoxia alters permeability to increasing ROS generation, which amplifies cytokine production. Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to hypoxia while secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1alpha, IL-6, and IL-8 was measured. IL-6 and IL-8 secretion increased fourfold over 24 h in a pattern corresponding to changes in HUVEC permeability measured by transendothelial electrical resistance (TEER). Addition of exogenous IL-6 to normoxic HUVEC monolayers caused time-dependent changes in TEER that mimicked the hypoxic response. An antibody to IL-6 significantly attenuated the hypoxia-induced changes in TEER (86 +/- 4 vs. 63 +/- 3% with hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect. To determine the role of hypoxia-induced ROS on this response, HUVEC monolayers were incubated with the antioxidants ebselen (50 microM) and N-acetyl-L-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 +/- 2 pg/ml with ebselen and 19 +/- 3 pg/ml with NAC vs. 140 +/- 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 +/- 2% with ebselen and 90 +/- 6% with NAC vs. 63 +/- 3% with hypoxia at 18 h). N-nitro-L-arginine (500 microM) did not decrease hypoxia-induced changes in dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS generated during hypoxia act as signaling elements, regulating secretion of the proinflammatory cytokines that lead to alterations of endothelial permeability.
Mechanical strain triggers a variety of cellular responses, but the underlying mechanotransduction process has not been established. Endothelial cells (EC) respond to mechanical strain by upregulating adhesion molecule expression through a signaling process involving reactive oxygen species (ROS), but the site of their generation is unknown. Mitochondria anchor to the cytoskeleton and could function as mechanotransducers by releasing ROS during cytoskeletal strain. In human umbilical vein EC (HUVEC), ROS production increased 221 +/- 17% during 6 h of cyclic strain vs. unstrained controls. Mitochondrial inhibitors diphenylene iodonium or rotenone abrogated this response, whereas inhibitors of nitric oxide (NO) synthase (L-nitroarginine), xanthine oxidase (allopurinol), or NAD(P)H oxidase (apocynin) had no effect. The antioxidants ebselen and diethyldithiocarbamate inhibited the increase in ROS, but the NO scavenger Hb had no effect. Thus strain induces ROS release from mitochondria. In other studies, HUVEC were rendered mitochondria deficient (rho0 EC) to determine the requirement for electron transport in the response to strain. Strain-induced 2'7'-dichlorofluorescein fluorescence was attenuated by >80% in rho0 EC compared with HUVEC (43 +/- 7 vs. 221 +/- 17%). Treatment with cytochalasin D abrogated strain-induced ROS production, indicating a requirement for the actin cytoskeleton. Cyclic strain (6 h) increased VCAM-1 expression in wild-type but not rho0 EC. Increases in NF-kappaB activation and VCAM-1 mRNA expression during strain were prevented by antioxidants. These findings demonstrate that mitochondria function as mechanotransducers in endothelium by increasing ROS signaling, which is required for strain-induced increase in VCAM-1 expression via NF-kappaB.
Abstract-Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2Ј,7Ј-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 mol/L) and diphenyleneiodonium (5 mol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-B (NF-B) activation, although they did not abrogate NF-B activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 mol/L), the xanthine oxidase inhibitor allopurinol (100 mol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 mol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-B activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia. These responses are essential for increases in the adhesion of polymorphonuclear leukocytes and the regulated increases in the trafficking of inflammatory cells across the endothelial barrier. Hypoxic stress often precedes or accompanies tissue inflammation, and previous studies have shown that tissue hypoxia can induce manifestations of the inflammatory response. In this regard, hypoxia augments the upregulation of endothelial cell surface receptors and the regulated increases in endothelial barrier permeability. 4 -6 However, the mechanistic link between cellular hypoxia and the initiation of these responses has not been clearly established. See page 525Recent studies have suggested that reactive oxygen species (ROS) may be important intracellular signaling messengers linking tissue hypoxia to the subsequent inflammatory responses. For example, in mesenteric venules of rats exposed to systemic hypoxia, Wood et al 7 found evidence of increased ROS generation that contributed to the stimulation of leukocyte-endothelial cell adhesion. We recently reported that ROS participate in the signaling responsible for the transient increase in en...
Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2Ј7Ј-dichlorofluorescin (DCFH), increased 152 Ϯ 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 M) or rotenone (2 M) attenuated this increase, whereas L-nitroarginine (100 M), allopurinol (100 M), or apocynin (30 M) had no effect. The antioxidants ebselen (5 M) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 M) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 M) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretchinduced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or Gö-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.reactive oxygen species; superoxide; cytoskeleton; cyclic stretch; protein kinase C ENDOTHELIAL DYSFUNCTION CONTRIBUTES to the pathogenesis in a wide range of diseases (9,20). In the lung, mechanical strain and shear stress represent normal stimuli to endothelial cells. However, excessive strain encountered during mechanical ventilation with large tidal volumes exacerbates lung injury and inflammation in patients with acute respiratory failure (7). An improved understanding of the mechanisms by which mechanical strain affects endothelial cell function could help to clarify how excessive lung strain exacerbates preexisting lung injury.Mechanical stretch has been shown to increase reactive oxygen species (ROS) production in endothelial cells, leading to the upregulation of cell adhesion molecules and chemokines (13, 51). Recent studies have implicated mitochondria as a source of ROS responsible for mediating flow-induced dilation in coronary arteries (32) and intercellular communication in vascular smooth muscle cells sub...
Many responses are initiated in cells subjected to mechanical deformation, including alterations in ion channel conductance, activation of signal transduction pathways, and altered expression of specific genes. Future progress in this field will require a critical distinction between cell systems that become activated during mechanical strain and the identity of the cellular mechanosensor that triggers subsequent responses.
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