Angiotensin II, NADPH oxidase, and redox signaling in the vasculature

Abstract

Significance: Angiotensin II (Ang II) influences the function of many cell types and regulates many organ systems, in large part through redox-sensitive processes. In the vascular system, Ang II is a potent vasoconstrictor and also promotes inflammation, hypertrophy, and fibrosis, which are important in vascular damage and remodeling in cardiovascular diseases. The diverse actions of Ang II are mediated via Ang II type 1 and Ang II type 2 receptors, which couple to various signaling molecules, including NADPH oxidase (Nox), which generates reactive oxygen species (ROS). ROS are now recognized as signaling molecules, critically placed in pathways activated by Ang II. Mechanisms linking Nox and Ang II are complex and not fully understood.

Recent advances: Ang II regulates vascular cell production of ROS through various recently characterized Noxs, including Nox1, Nox2, Nox4, and Nox5. Activation of these Noxs leads to ROS generation, which in turn influences many downstream signaling targets of Ang II, including MAP kinases, RhoA/Rho kinase, transcription factors, protein tyrosine phosphatases, and tyrosine kinases. Activation of these redox-sensitive pathways regulates vascular cell growth, inflammation, contraction, and senescence.

Critical issues: Although there is much evidence indicating a role for Nox/ROS in Ang II function, there is still a paucity of information on how Ang II exerts cell-specific effects through ROS and how Nox isoforms are differentially regulated by Ang II. Moreover, exact mechanisms whereby ROS induce oxidative modifications of signaling molecules mediating Ang II actions remain elusive.

Future directions: Future research should elucidate these issues to better understand the significance of Ang II and ROS in vascular (patho) biology.

FIG. 1.

Role of Nox-derived ROS in Ang II-mediated effects in vascular cells. Ang II binds to its AT₁R, which couples to heterometric Gq proteins, to activate PLC, leading to generation of IP3 and DAG, resulting in increased [Ca²⁺]_i that triggers phosphorylation of MLC₂₀ and stimulation of contraction. Ang II also induces contraction through the RhoA/Rho-kinase pathway that increases Ca²⁺ sensitivity by inhibiting MLCP. Ang II/AT₁R stimulates Nox-derived ROS formation, which regulates MAPKs, tyrosine kinases, PTPs, and transcription factors. Formation of ROS through Ang II/AT₁R further regulates AT₁R through a feed-forward mechanism (dashed lines). Ang II, angiotensin II; AT₁R, Ang II type 1 receptor; [Ca²⁺]_i, intracellular free-calcium concentration; DAG, diacylglycerol; IP3, inositol-3-phosphate; MAPKs, mitogen-activated protein kinases; MLCK, myosin light-chain kinase; MLCP, myosin light-chain phosphatase; Nox, NADPH oxidase; PLC, phospholipase C; PTPs, protein tyrosine phosphatases; ROS, reactive oxygen species.

FIG. 2.

Nox activation and differences between Nox homologs. Nox comprises a complex of membrane and cytosolic subunits. Nox2 is the classical prototype. Membrane proteins are p22phox and the Nox subunit, and form a noncovalent heterodimer. These proteins possess the electron transport apparatus and may act as a physical conduit for the electron transfer that occurs across the membrane. The cytosolic proteins (p47phox, p67phox, NoxO1, NoxA1, and rac 1/2) are cofactors for enzymatic activity and are used to initiate and/or regulate electron transfer. To Nox2 be activated, p47phox is phosphorylated and translocates from the cytosol to the membrane with the other cytosolic subunits (p67phox and rac1/2). Nox1 can also be activated in a similar way to Nox2, but possesses other cytosolic subunits such as NoxO1 and NoxA1. Nox4 does not require any cytosolic subunit to be activated. Nox4 is constitutively active in cells, and its activity is controlled by Poldip2. On the other hand, Nox5 activation is not dependent on any subunit, but because of calcium-binding domains (EF hands), its activity is controlled by calcium and calmodulin. NoxA1, NADPH activator 1; NoxO1, NADPH organizer 1; Poldip2, polymerase (DNA-directed) delta-interacting protein 2.

FIG. 3.

Ang II-stimulated redox-sensitive pathways that promote vascular remodeling. Generation of ROS by plasma membrane-associated Noxs and cytosolic Noxs in response to Ang II-AT₁R signaling leads to activation of multiple pathways that promote vascular injury and remodeling. e⁻, electron; NO, nitric oxide; SOD, superoxide dismutase.

FIG. 4.

Distribution of vascular Noxs in endothelial and VSMCs and downstream signaling pathways regulated by Nox-derived ROS. In basal conditions, Nox4 expression is greater in endothelial cells than in VSMCs. Nox1 and Nox2 are expressed in VSMCs from small arteries, whereas Nox1, but not Nox2, is expressed in VSMCs from large arteries. These Noxs are upregulated in pathological conditions. The exact distribution of vascular Nox isoforms in vivo still awaits confirmation. VSMCs, vascular smooth muscle cells.

FIG. 5.

Nox and eNOS by Ang II in endothelial cells. Ang II stimulates production of O₂^•− by Nox1, Nox2, and Nox5 through the AT₁R. Additionally, Ang II stimulates production of H₂O₂ directly through Nox4, and indirectly through the SOD-mediated conversion of O₂^•− produced by Nox1, 2, and 5. The O₂^•−-mediated oxidation and inactivation of the eNOS cofactor BH₄ promotes uncoupling of eNOS, leading to eNOS-mediated production of O₂^•−. Additionally, H₂O₂ inhibits DHFR, an enzyme that catalyzes the conversion of BH₂ to BH₄, which further reduces BH₄ bioavailability, leading to eNOS uncoupling. BH₂, dihydrobiopterin; BH₄, tetrahydrobiopterin; CaM, calmodulin; DHFR, dihydrofolate reductase; eNOS, endothelial nitric oxide synthase; Fe, eNOS heme domain; H₂O₂, hydrogen peroxide; O₂^•−, superoxide; p22, p22 phox (Nox subunit); p40, p40 phox (Nox subunit); p47, p47 phox (Nox subunit); p67, p67 phox (Nox subunit); Rac, Rho-like GTPase Rac.

FIG. 6.

Ang II-mediated redox-sensitive growth signaling in VSMCs. In VSMCs, binding of Ang II to the AT₁R leads to increased ROS generation, in part, via activation of c-Src-induced Nox activation. ROS stimulate nonreceptor tyrosine kinases such as FAK, JAK and PI3K, and PLC/PKC as well as receptor tyrosine kinases, such as EGFR, IGFR, and PDGFR. Redox signaling in turn regulates downstream MEK cascades, leading to phosphorylation of MAPKs, which induce growth, apoptosis, differentiation, migration, and inflammation of VSMCs. Ang II/AT₁R also induces MMP-mediated extracellular release of HB-EGF, which then stimulates EGFR transactivation and ERK1/2 MAPK activation. EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; HB-EGF, heparin-binding epidermal growth factor; IGFR, insulin-like growth factor receptor; JAK, Janus-activated kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/ERK kinase; MMP, matrix metalloproteinase; p, phosphorylation; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PLC, phospholipase C; STAT, signal transducers and activators of transcription.