Haruhiko Ohtsu

Angiotensin II regulates vascular and endothelial dysfunction: recent topics of Angiotensin II type-1 receptor signaling in the vasculature.

Accumulating evidence strongly implicates angiotensin II (AngII) intracellular signaling in mediating cardiovascular diseases such as hypertension, atherosclerosis and restenosis after vascular injury. In vascular smooth muscle cells (VSMCs), through its G-protein-coupled AngII Type 1 receptor (AT(1)), AngII activates various intracellular protein kinases, such as receptor or non-receptor tyrosine kinases, which includes epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), c-Src, PYK2, FAK, JAK2. In addition, AngII activates serine/threonine kinases such as mitogen-activated protein kinase (MAPK) family, p70 S6 kinase, Akt/protein kinase B and various protein kinase C isoforms. In VSMCs, AngII also induces the generation of intracellular reactive oxygen species (ROS), which play critical roles in activation and modulation of above signal transduction. Less is known about endothelial cell (EC) AngII signaling than VSMCs, however, recent studies suggest that endothelial AngII signaling negatively regulates the nitric oxide (NO) signaling pathway and thereby induces endothelial dysfunction. Moreover, in both VSMCs and ECs, AngII signaling cross-talk with insulin signaling might be involved in insulin resistance, an important risk factor in the development of cardiovascular diseases. In fact, clinical and pharmacological studies showed that AngII infusion induces insulin resistance and AngII converting enzyme inhibitors and AT(1) receptor blockers improve insulin sensitivity. In this review, we focus on the recent findings that suggest the existence of novel signaling mechanisms whereby AngII mediates processes, such as activation of receptor or non-receptor tyrosine kinases and ROS, as well as cross-talk between insulin and NO signal transduction in VSMCs and ECs.

Signal-Crosstalk Between Rho/ROCK and c-Jun NH2-Terminal Kinase Mediates Migration of Vascular Smooth Muscle Cells Stimulated by Angiotensin II

Background—Rho and its effector Rho-kinase/ROCK mediate cytoskeletal reorganization as well as smooth muscle contraction. Recent studies indicate that Rho and ROCK are critically involved in vascular remodeling. Here, we tested the hypothesis that Rho/ROCK are critically involved in angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs) by mediating a specific signal cross-talk. Methods and Results—Immunoblotting demonstrated that Ang II stimulated phosphorylation of a ROCK substrate, regulatory myosin phosphatase targeting subunit (MYPT)-1. Phosphorylation of MYPT-1 as well as migration of VSMCs induced by Ang II was inhibited by dominant-negative Rho (dnRho) or ROCK inhibitor, Y27632. Ang II–induced c-Jun NH2-terminal kinase (JNK) activation, but extracellular signal-regulated kinase (ERK) activation was not mediated through Rho/ROCK. Thus, infection of adenovirus encoding dnJNK inhibited VSMC migration by Ang II. We have further demonstrated that the Rho/ROCK activation by Ang II requires protein kinase C-&dgr; (PKC&dgr;) and proline-rich tyrosine kinase 2 (PYK2) activation, but not epidermal growth factor receptor transactivation. Also, VSMCs express PDZ-Rho guanine nucleotide exchange factor (GEF) and Ang II stimulated PYK2 association with tyrosine phosphorylated PDZ-RhoGEF. Conclusions—PKC&dgr;/PYK2-dependent Rho/ROCK activation through PDZ-RhoGEF mediates Ang II–induced VSMC migration via JNK activation in VSMCs, providing a novel mechanistic role of the Rho/ROCK cascade that is involved in vascular remodeling.

ADAM17 Mediates Epidermal Growth Factor Receptor Transactivation and Vascular Smooth Muscle Cell Hypertrophy Induced by Angiotensin II

Background—Angiotensin II (Ang II) promotes growth of vascular smooth muscle cells (VSMCs) via epidermal growth factor (EGF) receptor (EGFR) transactivation mediated through a metalloprotease-dependent shedding of heparin-binding EGF-like growth factor (HB-EGF). However, the identity of the metalloprotease responsible for this process remains unknown. Methods and Results—To identify the metalloprotease required for Ang II-induced EGFR transactivation, primary cultured aortic VSMCs were infected with retrovirus encoding dominant negative (dn) mutant of ADAM10 or ADAM17. EGFR transactivation induced by Ang II was inhibited in VSMCs infected with dnADAM17 retrovirus but not with dnADAM10 retrovirus. However, Ang II comparably stimulated intracellular Ca2+ elevation and JAK2 tyrosine phosphorylation in these VSMCs. In addition, dnADAM17 inhibited HB-EGF shedding induced by Ang II in A10 VSMCs expressing the AT1 receptor. Moreover, Ang II enhanced protein synthesis and cell volume in VSMCs infected with control retrovirus, but not in VSMCs infected with dnADAM17 retrovirus. Conclusion—ADAM17 activated by the AT1 receptor is responsible for EGFR transactivation and subsequent protein synthesis in VSMCs. These findings demonstrate a previously missing molecular mechanism by which Ang II promotes vascular remodeling.

Central role of Gq in the hypertrophic signal transduction of angiotensin II in vascular smooth muscle cells.

The angiotensin II (AngII) type 1 receptor (AT(1)) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that G(q) is the major G protein activated by the AT(1) receptor, the requirement of G(q) for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of G(q) for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca(2+) elevation was completely inhibited by a pharmacological G(q) inhibitor as well as by adenovirus encoding a G(q) inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The G(q) inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT(1) agonist did not stimulate these hypertrophic responses. In conclusion, G(q) appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular G(q) seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.

Novel Role of Protein Kinase C-δ Tyr311 Phosphorylation in Vascular Smooth Muscle Cell Hypertrophy by Angiotensin II

We have shown previously that activation of protein kinase C-&dgr; (PKC&dgr;) is required for angiotensin II (Ang II)–induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC&dgr; phosphorylation at Tyr311 plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC&dgr; phosphorylation at Tyr311, and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC&dgr; was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr311 by Ang II. This phosphorylation was markedly blocked by an Src family kinase inhibitor and dominant-negative Src but not by an epidermal growth factor receptor kinase inhibitor. Ang II-induced Akt phosphorylation and hypertrophic responses were significantly enhanced in VSMCs expressing PKC&dgr; wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC&dgr; Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC&dgr; K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC&dgr; kinase activation but also the Src-dependent Tyr311 phosphorylation contributes to Akt activation and subsequent VSMC hypertrophy induced by Ang II, thus signifying a novel molecular mechanism for enhancement of cardiovascular diseases induced by Ang II.