Kaikobad Irani

Oxidant Signaling in Vascular Cell Growth, Death, and Survival A Review of the Roles of Reactive Oxygen Species in Smooth Muscle and Endothelial Cell Mitogenic and Apoptotic Signaling

Reactive oxygen species (ROS) have been traditionally regarded as toxic byproducts of aerobic metabolism. However, ROS can also act as intracellular signaling molecules in vascular cells. ROS can mediate phenotypes in vascular endothelial and smooth muscle cells that may be considered both physiological and pathophysiological. Among these are growth, apoptosis, and survival. The specific response elicited by reactive oxygen intermediaries is determined by their specific intracellular target(s). This, in turn, is dependent on the species of oxidant(s) produced, the source and therefore subcellular localization of the oxidant(s), the kinetics of production, and the quantities produced. A fuller understanding of how ROS regulate mitogenesis and apoptosis in vascular smooth muscle and endothelial cells will permit the development of novel strategies to modify or prevent vascular diseases in which these phenotypes predominate.

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)

Protection from reoxygenation injury by inhibition of rac1.

We demonstrate that adenoviral-mediated gene transfer of a dominant negative rac1 gene product (N17rac1) inhibits the intracellular burst of reactive oxygen species (ROS) that occurs after reoxygenation of vascular smooth muscle cells. In contrast, expression of a dominant negative ras gene (N17ras) had no effect. Challenge of control cells and cells expressing N17rac1 with a direct oxidant stress produced an equivalent increase in intracellular ROS levels and subsequent cell death. This suggests that N17rac1 expression appears to block production of harmful oxygen radicals and does not act directly or indirectly to scavenge ROS generated during reoxygenation. Expression of N17rac1 results in protection from hypoxia/reoxygenation-induced cell death in a variety of cell types including vascular smooth muscle cells, fibroblasts, endothelial cells, and ventricular myocytes. These results suggest that reoxygenation injury requires the activation of rac proteins, and that inhibition of rac-dependent pathways may be a useful strategy for the prevention of reperfusion injury in ischemic tissues.

A requirement for the rac1 GTPase in the signal transduction pathway leading to cardiac myocyte hypertrophy.

We have used adenoviral-mediated gene transfer of a constitutively active (V12rac1) and dominant negative (N17rac1) isoform of rac1 to assess the role of this small GTPase in cardiac myocyte hypertrophy. Expression of V12rac1 in neonatal cardiac myocytes results in sarcomeric reorganization and an increase in cell size that is indistinguishable from ligand-stimulated hypertrophy. In addition, V12rac1 expression leads to an increase in atrial natriuretic peptide secretion. In contrast, expression of N17rac1, but not a truncated form of Raf-1, attenuated the morphological hypertrophy associated with phenylephrine stimulation. Consistent with the observed effects on morphology, expression of V12rac1 resulted in an increase in new protein synthesis, while N17rac1 expression inhibited phenylephrine-induced leucine incorporation. These results suggest rac1 is an essential element of the signaling pathway leading to cardiac myocyte hypertrophy.

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.

Alterations in the Expression of the Apurinic/Apyrimidinic Endonuclease-1/redox Factor-1 (APE/Ref-1) in Human Melanoma and Identification of the Therapeutic Potential of Resveratrol as an APE/Ref-1 Inhibitor

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein involved in DNA base excision repair and redox regulation of many transcription factors. In different melanoma cell lines, we found that both nucleus and cytoplasm exhibited higher levels of Ref-1 compared with normal melanocytes. Similar increases of Ref-1 expression, detected by immunohistofluorescence, were also evident in nevi and malignant melanoma biopsies compared with normal skin, which were predominantly localized in the nucleus. Using recombinant adenovirus Adref-1, encoding full-length Ref-1, we transiently overexpressed APE/Ref-1 in human melanocytes, which protected these cells from UVB-induced apoptosis and increased foci formation in culture. Ref-1 overexpression also protected melanoma cells from cisplatin- or H2O2-induced apoptosis, whereas increased apoptosis was observed with Ref-1 antisense construct infection. These observations suggested that intracellular Ref-1 levels played an important role in sensitization of melanoma cells to apoptosis. Electrophoretic mobility shift assay results showed that in both cultured primary and metastatic melanomas DNA-binding activities of activator protein-1 and nuclear factor-κB were significantly diminished or shifted when anti-APE/Ref-1 antibody was added to deplete APE/Ref-1 from the binding complexes. Induced nuclear factor-κB transcriptional activities were also evident after Ref-1 overexpression. Furthermore, using three-dimensional molecular structure modeling and virtual screening, we found that resveratrol, a natural compound found in fruits and vegetables, docks into a druggable pocket of Ref-1 protein. In vitro studies revealed that resveratrol inhibited, in a dose-dependent manner, Ref-1-activated activator protein-1 DNA-binding activities as well as Ref-1 endonuclease activities and rendered melanoma cells more sensitive to dacarbazine treatment. [Mol Cancer Ther 2005;4(12):1923–35]

Redox factor-1: an extra-nuclear role in the regulation of endothelial oxidative stress and apoptosis

The rac1 GTPase promotes oxidative stress through reactive oxygen species (ROS) production, whereas the DNA repair enzyme and transcriptional regulator redox factor-1 (ref-1) protects against cell death due to oxidative stimuli. However, the function of ref-1 in regulating intracellular oxidative stress, particularly that induced by rac1, has not been defined. We examined the role of ref-1 in vascular endothelial cell oxidative stress and apoptosis. Ref-1 was expressed in both the cytoplasm and nuclei of resting endothelial cells. Cytoplasmic ref-1 translocated to the nucleus with the oxidative trigger hypoxia/reoxygenation (H/R). Forced cytoplasmic overexpression of ref-1 suppressed H/R-induced oxidative stress (H2O2 production), NF-κB activation, and apoptosis, and also mitigated rac1-regulated H2O2 production and NF-κB transcriptional activity. We conclude that inhibition of oxidative stress is another mechanism by which ref-1 protects against apoptosis, and that this is achieved through modulation of cytoplasmic rac1-regulated ROS generation. This suggests a novel extra-nuclear function of ref-1.