Shailesh S. Deshpande

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

Reactive oxygen species (ROS) have been implicated as mediators of tumor necrosis factor‐alpha (TNF) ‐induced apoptosis. In addition to leading to cell death, ROS can also promote cell growth and/or survival. We investigated these two roles of ROS in TNF‐induced endothelial cell apoptosis. Human umbilical vein endothelial cells (HUVECs) stimulated with TNF produced an intracellular burst of ROS. Adenoviral‐mediated gene transfer of a dominant negative form of the small GTPase Rac1 (Rac1N17) partially suppressed the TNF‐induced oxidative burst without affecting TNF‐induced mitochondrial ROS production. HUVECs were protected from TNF‐induced apoptosis. Expression of Rac1N17 blocked TNF‐induced activation of nuclear factor‐kappa B (NF‐κB), increased activity of caspase‐3, and markedly augmented endothelial cell susceptibility to TNF‐induced apoptosis. Direct inhibition of NF‐κB through adenoviral expression of the super repressor form of inhibitor of kBα (I‐κB S32/36A) also increased susceptibility of HUVECs to TNF‐induced apoptosis. Rotenone, a mitochondrial electron transport chain inhibitor, suppressed TNF‐induced mitochondrial ROS production, proteolytic cleavage of procaspase‐3, and apoptosis. These findings show that Rac1 is an important regulator of TNF‐induced ROS production in endothelial cells. Moreover, they suggest that Rac1‐dependent ROS, directly or indirecly, lead to protection against TNF‐induced death, whereas mitochondrial‐derived ROS promote TNF‐induced apoptosis.—Deshpande, S. S., Angkeow, P., Huang, J., Ozaki, M., Irani, K. Rac1 inhibits TNF‐α‐induced endothelial cell apoptosis: dual regulation by reactive oxygen species. FASEB J. 14, 1705–1714 (2000)

Vascular endothelial growth factor induces manganese-superoxide dismutase expression in endothelial cells by a Rac1-regulated NADPH oxidase-dependent mechanism

Vascular endothelial growth factor (VEGF) is a potent vascular endothelial cell‐specific mitogen that modulates endothelial cell function. In the present study, we show that VEGF induces manganese‐superoxide dismutase (MnSOD) mRNA and protein in human coronary artery endothelial cells (HCAEC) and pulmonary artery endothelial cells. VEGF‐mediated induction of MnSOD mRNA was inhibited by pretreatment with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI), and 4‐(2‐aminoethyl)‐benzenesulfonyl fluoride, but not with the nitric oxide synthase inhibitor L‐NAME (N‐monomethyl‐L‐arginine) or the xanthine oxidase inhibitor allopurinol. VEGF stimulation of MnSOD was also inhibited by adenoviral‐mediated overexpression of catalase Cu, Zn‐SOD and a dominant‐negative form of the small GTPase component of NADPH oxidase Rac1 (Rac1N17). Treatment of HCAEC with VEGF resulted in a transient increase in ROS production at 20 min, as measured by 2′,7′‐dichlorodihydrofluorescein oxidation. This effect was abrogated by expression of Rac1N 17. Taken together, these findings suggest that VEGF induces MnSOD by an NADPH oxidase‐dependent mechanism and that VEGF signaling in the endothelium is coupled to the redox state of the cell.

Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo

Reperfusion of ischemic tissue results in the generation of reactive oxygen species that contribute to tissue injury. The sources of reactive oxygen species in reperfused tissue are not fully characterized. We hypothesized that the small GTPase Rac1 mediates the oxidative burst in reperfused tissue and thereby contributes to reperfusion injury. In an in vivo model of mouse hepatic isch‐emia/reperfusion injury, recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion‐induced production of reactive oxygen species and lipid peroxides, activation of nuclear factor‐kappa B, and resulted in a significant reduction of acute liver necrosis. Expression of Rac1N17 also suppressed ischemia/reperfusion‐induced acute apoptosis. The protection offered by Rac1N17 was also evident in knockout mice deficient for the gp91phox component of the phagocyte NADPH oxidase. This work demonstrates the crucial role of a Rac1‐regulated oxidase in mediating the production of injurious reactive oxygen species, which contribute to acute necrotic and apoptotic cell death induced by isch‐emia/reperfusion in vivo. Targeted inhibition of this oxidase, which is distinct from the phagocyte NADPH oxidase, should provide a new avenue for in vivo therapy aimed at protecting organs at risk from ischemia/reperfusion injury.—Ozaki, M., Deshpande, S. S., Angkeow, P., Bellan, J., Lowenstein, C. J., Dinauer, M. C., Goldschmidt‐Clermont, P. J., Irani, K. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion‐induced necrosis and apoptosis in vivo. FASEB J. 14, 418—429 (2000)

Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2 for 5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.The shear-induced intracellular signal transduction pathway in vascular endothelial cells involves tyrosine phosphorylation and activation of mitogen-activated protein (MAP) kinase, which may be responsible for the sustained release of nitric oxide. MAP kinase is known to be activated by reactive oxygen species (ROS), such as H2O2, in several cell types. ROS production in ligand-stimulated nonphagocytic cells appears to require the participation of a Ras-related small GTP-binding protein, Rac1. We hypothesized that Rac1 might serve as a mediator for the effect of shear stress on MAP kinase activation. Exposure of bovine aortic endothelial cells to laminar shear stress of 20 dyn/cm2 for 5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited in a dose-dependent manner the shear-stimulated increase in total cytosolic and, specifically, MAP kinase tyrosine phosphorylation. Hence, the onset of shear stress caused an enhanced generation of intracellular ROS, as evidenced by an oxidized protein detection kit, which were required for the shear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing a dominant negative Rac1 gene product (N17rac1). We concluded that the GTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAP kinase via regulation of the flow-dependent redox changes in endothelial cells in physiological and pathological circumstances.

[Ca2+] i Oscillation Frequency Regulates Agonist-stimulated NF-κB Transcriptional Activity

In nonexcitable cells, stimulation by high agonist concentrations typically produces a biphasic increase in cytosolic Ca2+ ([Ca2+] i ). This response is characterized by a transient initial increase because of intracellular Ca2+ release followed by a sustained elevation which varies in amplitude depending on the nature of the stimulus. In contrast, low-level stimulation often evokes oscillatory changes in [Ca2+] i . The specific information provided by repetitive [Ca2+] i spikes appears to be encoded in the frequency rather than in the amplitude of [Ca2+] i oscillations. The specific, membrane-permeable inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) receptor blocker Xestospongin C (XeC, 2–20 μm) was used to affect [Ca2+] i signaling in human aortic endothelial cells (HAEC) during an established response to low-level (1 μm) histamine stimulation. XeC produced a dose-dependent decrease in the frequency of [Ca2+] i oscillations during histamine stimulation without affecting oscillation amplitude. Histamine stimulated a 14-fold increase in NF-κB-chloramphenicol acetyltransferase reporter gene activity that was dose-dependently decreased by XeC. Thus, during low-level agonist stimulation, [Ca2+] i oscillation frequency regulates nuclear transcription in HAEC.

Wall Stiffness Suppresses Akt/eNOS and Cytoprotection in Pulse-Perfused Endothelium

Abstract—Increased steady shear stress stimulates nitric oxide synthase (eNOS) in part by Akt-dependent phosphorylation. Arteries in vivo are exposed to pulse perfusion (PP) combining phasic shear with stretch. In compliant vessels, enhancing PP lowers vascular tone by stimulating eNOS; whereas in aged, stiff arteries, flow-mediated dilation declines and PP is a prominent risk factor. Here, we tested the hypothesis that reduced wall distensibility alters PP-induced eNOS/Akt mechano-signaling. Bovine aortic endothelial cells cultured within distensible tubes were exposed to physiological nonreversing steady or PP (7 dynes/cm2 mean shear, pulse pressure 0 or 90 mm Hg×2 hours) in a custom servo-system. In compliant tubes, PP doubled Akt phosphorylation above nonpulsatile flow levels, whereas P-Akt declined to static levels from PP in stiffer tubes. eNOS phosphorylation (S-1179) similarly increased with PP in compliant tubes but was nearly undetectable with increased PP in stiffer tubes. After PP, brief exposure of cells to ultraviolet irradiation (oxidant stress) and subsequent culture revealed cytoprotection in compliant tubes but diffuse cytotoxicity and cell detachment in stiffer tubes. NOS inhibition by L-NAME converted compliant-tube post-UV behavior to that of stiffer tubes. These data provide novel evidence that wall compliance can directionally mediate endothelial Akt/eNOS phosphorylation and mechano-signaling. This may help explain increased vascular risks resulting from artery stiffening.

NADPH Oxidase Activation Increases the Sensitivity of Intracellular Ca2+ Stores to Inositol 1,4,5-Trisphosphate in Human Endothelial Cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca2+, but whether NADPH oxidase activation directly affects Ca2+ signaling is unknown. NADPH stimulated the production of superoxide anion and H2O2 in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H2O2 each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP3) required to release intracellularly stored Ca2+ and shifted the InsP3-Ca2+ release dose-response curve to the left. Concentrations of H2O2 as low as 3 μm increased the sensitivity of intracellular Ca2+ stores to InsP3 and decreased the InsP3 EC50 from 423.2 ± 54.9 to 276.9 ± 14.4 nm. The effect of NADPH on InsP3-stimulated Ca2+ release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H2O2 increases the sensitivity of intracellular Ca2+ stores to InsP3 in human endothelial cells. Since Ca2+-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.

Rac1 regulates stress-induced, redox-dependent heat shock factor activation.

The signaling pathway by which environmental stresses activate heat shock factors (HSFs) is not completely understood. We show that the small GTPase rac1, and Rac1-regulated reactive oxygen species (ROS) play an important role in stress-stimulated heat shock response. A dominant-negative allele of Rac1 (Rac1N17) inhibits the hypoxia/reoxygenation and sodium arsenite-induced transcriptional activity of HSF-1 and the transcription of heat shock protein 70. Rac1N17 also suppresses the production of intracellular ROS induced by hypoxia/reoxygenation or sodium arsenite. Moreover, direct suppression of intracellular ROS levels by antioxidants decreases stress-stimulated HSF activity. However, expression of a constitutively active mutant of Rac1 (Rac1V12) in the absence of extracellular stresses does not increase intracellular ROS levels or induce the heat shock response. These results show that Rac1 is a necessary but insufficient component of the stress-induced signaling pathway that leads to ROS production, activation of HSFs, and transcription of heat shock proteins.

Expression of Id1 Results in Apoptosis of Cardiac Myocytes through a Redox-dependent Mechanism

We have constructed a recombinant adenovirus (Ad.Id1) that allows for efficient expression of the helix-loop-helix protein Id1. After infection with Ad.Id1, neonatal cardiac myocytes display a significant reduction in viability, which was proportional to the level of Id1 expression. A similar effect was observed in adult myocytes. Morphological and biochemical assays demonstrated that Id1 expression resulted in myocyte apoptosis. In contrast, expression of Id1 in endothelial cells, vascular smooth muscle cells, or fibroblasts did not affect the viability of these cells. Along with the induction of apoptosis, the expression of Id1 in neonatal cardiac myocytes resulted in an increase in the level of intracellular reactive oxygen species. The source of these reactive oxygen species appears to be the mitochondria. Reducing the ambient oxygen concentration or treatment with a cell-permeant H2O2 scavenger prevented Id1-stimulated apoptosis in cardiac myocytes. These results suggest that the expression of Id1 leads to the induction of apoptosis in cardiac myocytes through a redox-dependent mechanism.

Constitutive Activation of rac1 Results in Mitochondrial Oxidative Stress and Induces Premature Endothelial Cell Senescence

Objective—Oxidative stress has been implicated in cellular senescence and vascular aging. We determined the role and mechanism of the small GTPase rac1 in vascular endothelial cell senescence. Methods and Results—Adenoviral-mediated expression of the constitutively active allele of rac 1 (rac 1V12) in human umbilical vein endothelial cells resulted in mitochondrial oxidative stress with induction of biochemical, molecular, and morphological features of senescence. Suppression of mitochondrial oxidative stress abrogated rac 1-induced premature senescence. Rac 1V12 expression also resulted in an increase in endothelial ceramide levels. Moreover, premature endothelial cell senescence induced by an exogenous cell-permeable ceramide analog was not suppressed by inhibiting endogenous rac 1 signaling. Finally, in human umbilical vein endothelial cells that had undergone replicative senescence, rac 1 was not activated, and expression of the dominant-negative rac 1 allele (rac 1N17) did not suppress mitochondrial oxidative stress. Conclusions—These findings paint a picture in which the constitutive activation of rac 1, via the generation of ceramide, results in mitochondrial oxidative stress and premature endothelial cell senescence. However, they speak against a role for endogenous rac1 activation in the induction of mitochondrial oxidative stress associated with replicative senescence of endothelial cells.