Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells

doi:10.1016/j.vph.2005.05.002

Vascular Pharmacology

Volume 43, Issue 3, September 2005, Pages 148-156

https://doi.org/10.1016/j.vph.2005.05.002 Get rights and content

Abstract

Both in vivo models of ischemia/reperfusion and in vitro models of hypoxia (H)/reoxygenation (R) have demonstrated the crucial role of the Rac1-regulated NADPH oxidase in the production of injurious reactive oxygen species (ROS) by vascular endothelial cells (ECs). Since membrane lipid peroxidation has been established as one of the mechanisms leading to cell death, we examined lipid peroxidation in H/R-exposed cultured human umbilical vein ECs (HUVECs) and the role of Rac1 in this process. H (24 h at 1% O₂)/R (5 min) caused an increase in intracellular ROS production compared to a normoxic control, as measured by dichlorofluorescin fluorescence. Nutrient deprivation (ND; 24 h), a component of H, was sufficient to induce a similar increase in ROS under normoxia. Either H(24 h)/R (2 h) or ND (24 h) induced increases in lipid peroxidation of similar magnitude as measured by flow cytometry of diphenyl-1-pyrenylphosphine-loaded HUVECs and Western blotting analysis of 4-hydroxy-2-nonenal-modified proteins in cell lysates. In cells infected with a control adenovirus, H (24 h)/R (2 h) and ND (24 h) resulted in increases in NADPH-dependent superoxide production by 5- and 9-fold, respectively, as measured by lucigenin chemiluminescence. Infection of HUVECs with an adenovirus that encodes the dominant-negative allele of Rac1 (Rac1N17) abolished these increases. Rac1N17 expression also suppressed the H/R- and ND-induced increases in lipid peroxidation. In conclusion, ROS generated via the Rac1-dependent pathway are major contributors to the H/R-induced lipid peroxidation in HUVECs, and ND is able to induce Rac1-dependent ROS production and lipid peroxidation of at least the same magnitude as H/R.

Introduction

Reperfusion of ischemic tissue results in the generation of reactive oxygen species (ROS) that contribute to tissue injury (Granger and Korthuis, 1995, Grisham et al., 1998, Zweier et al., 1988). Earlier studies suggested that xanthine oxidase and the mitochondrial electron transport chain are important sources of ROS produced during reperfusion (Acosta and Li, 1979, Ratych et al., 1987, Russell et al., 1994). However, ROS production by the isolated and continuously ventilated ischemic mouse lungs was shown to originate from the membrane-associated NADPH oxidase complex of vascular endothelial cells (ECs) (Al-Mehdi et al., 1998). ROS production by the membrane-bound NADPH oxidase in both phagocytic and nonphagocytic cells is regulated by Rac1, a member of the Rho family of small GTPases (Abo et al., 1991). The crucial role of the Rac1-regulated NADPH oxidase in generating the injurious ROS was demonstrated in an in vivo model of mouse hepatic ischemia/reperfusion injury, in which the recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion-induced ROS production and lipid peroxidation and significantly reduced apoptosis and necrosis in the liver (Ozaki et al., 2000).

In in vitro models of ischemia/reperfusion, injury due to exposure of cultured ECs to hypoxia (H)/reoxygenation (R) was mediated by endogenous ROS production and was a function of severity of H and duration of both H and R (McLeod and Sevanian, 1997, Michiels et al., 1992, Terada, 1996). As in the in vivo models, Rac1N17 expression in cultured human umbilical vein ECs (HUVECs) inhibited the H/R-induced burst in ROS generation and significantly reduced cell death, as compared to control cells (Kim et al., 1998). Peroxidation of the phospholipids in the plasma membrane mediated by ROS has been established as one of the mechanisms of cell death (Girotti, 1998). Phospholipid peroxidation perturbs the integrity of the lipid bilayer of cell membranes leading to increased plasma membrane permeability and apoptotic cell death (Kriska et al., 2002, Pak et al., 2002). Exposure of cultured ECs to H/R was shown to increase both the extent of lipid peroxidation and the number of apoptotic cells (Li et al., 1998, McLeod and Alayash, 1999, Yang et al., 2001). However, the effect of Rac1 that regulates the NADPH oxidase-mediated generation of ROS on the membrane lipid peroxidation in ECs exposed to H/R has not been reported.

Therefore, in the present study, we investigated the effect of H/R exposure on lipid peroxidation in cultured HUVECs and the role of Rac1 in this process. Cells were exposed to severe prolonged H (24 h at 1% O₂) followed by R up to 2 h. During exposure to H, HUVECs were maintained in serum-free media, so that they were also subjected to nutrient deprivation (ND), to create H more physiologically relevant to ischemia. Since a recent report has revealed that ND alone is sufficient to induce production of monocyte chemoattractant protein (MCP)-1 in ECs that requires ROS generated by the Rac1-dependent pathway (Lopes et al., 2002), we also investigated the effect of ND (24 h) on lipid peroxidation in cultured HUVECs infected either with control adenovirus or with adenovirus encoding Rac1N17. The extent of lipid peroxidation in HUVECs was determined by flow-cytometric analysis of the intracellular formation of lipid hydroperoxides using diphenyl-1-pyrenylphosphine (DPPP), and by Western blotting analysis of the formation of 4-hydroxy-2-nonenal (HNE)-modified proteins in cell lysates using anti-HNE polyclonal antibodies. Intracellular generation of ROS was determined by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)-coupled flow cytometry. Cellular NADPH oxidase activity was determined by lucigenin chemiluminescence assay. Our results showed that H (24 h)/R (5 min–2 h) induces ROS production and lipid peroxidation mediated by the Rac1-regulated NADPH oxidase in HUVECs. Our results further revealed that ND (24 h) is sufficient to induce lipid peroxidation of similar magnitude as compared to H (24 h)/R (2 h), which is partially due to Rac1-mediated ROS production.

Section snippets

Reagents

tert-Butyl hydroperoxide (tBOOH), cumene hydroperoxide (CumOOH), hydrogen peroxide (H₂O₂), lucigenin, NADPH, and recombinant (Escherichia coli) human tumor necrosis factor (TNF)-α were purchased from Sigma Chemical Company (St. Louis, MO). DCFH-DA and DPPP were purchased from Molecular Probes (Eugene, OR). Stock reagents were prepared in medium M199, except for DPPP that was dissolved in dimethylsulfoxide (DMSO) at a concentration of 5 mM. Rabbit anti-HNE antiserum was from Alpha Diagnostic

Both H/R and ND increase EC ROS production

ROS generation in HUVECs was monitored using DCFH-DA and flow cytometry. Exogenously added H₂O₂, and organic hydroperoxides (tBOOH and CumOOH) were chosen as positive controls to show an increase in intracellular ROS at concentrations and times of incubation that were previously shown to induce lipid peroxidation in cell membranes (Okimoto et al., 2000, Takahashi et al., 2001). From the flow cytometry histograms, it was found that H₂O₂ (0.5 or 5 μM), tBOOH (0.75 or 1.5 μM) and CumOOH (1 or 4

Discussion

The present study provides evidence that targeted inhibition of the small GTPase Rac1, which regulates ROS production via a membrane-bound NADPH oxidase (Abo et al., 1991), protects against the H/R-induced lipid peroxidation in cultured HUVECs. This agrees with findings from in vivo studies which have revealed that ischemia-induced lipid peroxidation in perfused rat lungs, as measured by thiobarbituric acid reactive substances, was found significantly inhibited by pretreatment with PR-39, a

Acknowledgements

We thank K. Irani for the generous gift of AdβGal and AdRac1N17, and A. Kolmakova and S. Deshpande for technical assistance. This work was supported by National Institutes of Health grant RO1-HL67027 (B. R. Alevriadou). S. F. Martin was partially supported by a fellowship from Ministerio de Educación y Cultura, Spain.

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Rac1 inhibition protects against hypoxia/reoxygenation-induced lipid peroxidation in human vascular endothelial cells

Abstract

Introduction

Section snippets

Reagents

Both H/R and ND increase EC ROS production

Discussion

Acknowledgements

Free Radic. Biol. Med.

Free Radic. Biol. Med.

J. Lipid Res.

Free Radic. Biol. Med.

J. Immunol. Methods

Methods Enzymol.

J. Biol. Chem.

Free Radic. Biol. Med.

Free Radic. Biol. Med.

FEBS Lett.

J. Biol. Chem.

Free Radic. Biol. Med.

Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1

Nature

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

In Vitro

Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+

Circ. Res.

Rac1 inhibits TNF-alpha-induced endothelial cell apoptosis: dual regulation by reactive oxygen species

FASEB J.

Physiologic mechanisms of post-ischemic tissue injury. [Review]

Annu. Rev. Physiol.

In vivo suppression of injury-induced vascular smooth muscle cell accumulation using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene

Proc. Natl. Acad. Sci. U. S. A.

4-Hydroxynonenal induces dysfunction and apoptosis of cultured endothelial cells

J. Cell. Physiol.

Apoptosis in vascular endothelial cells caused by serum deprivation, oxidative stress and transforming growth factor-β

Endothelium

Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K⁺