ssDNA-dsRNAs are cleaved at the next to its chimera-junction point by an unknown RNase activity.

We found that there is an unknown aspect in serum RNases that cleaves ssDNA-dsRNA and ssRNA-dsRNA. In the first step, RNase cleaves the phosphodiester linkage between the first and second RNA, where the first one is connected to the single stranded RNA or DNA. In the second step, the ssRNA overhang attached siRNA is cleaved. When the 2' hydroxyl of the first RNA was replaced with methoxy, the cleavage did not occur. This RNase activity can be considered related to defense system against exogenous genetic materials.

Endothelial nitric oxide synthase inhibits G12/13 and rho-kinase activated by the angiotensin II type-1 receptor: implication in vascular migration.

BACKGROUND: Although, endothelial nitric oxide (NO) synthase (eNOS) is believed to antagonize vascular remodeling induced by the angiotensin II (AngII) type-1 receptor, the exact signaling mechanism remains unclear. METHODS AND RESULTS: By expressing eNOS to vascular smooth muscle cells (VSMCs) via adenovirus, we investigated a signal transduction mechanism of the eNOS gene transfer in preventing vascular remodeling induced by AngII. We found marked inhibition of AngII-induced Rho/Rho-kinase activation and subsequent VSMC migration by eNOS gene transfer whereas G(q)-dependent transactivation of the epidermal growth factor receptor by AngII remains intact. This could be explained by the specific inhibition of G(12/13) activation by eNOS-mediated G(12/13) phosphorylation. CONCLUSIONS: The eNOS/NO cascade specifically targets the Rho/Rho-kinase system via inhibition of G(12/13) to prevent vascular migration induced by AngII, representing a novel signal cross-talk in cardiovascular protection by NO.

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.

Angiotensin II signal transduction through the AT1 receptor: novel insights into mechanisms and pathophysiology.

The intracellular signal transduction of AngII (angiotensin II) has been implicated in cardiovascular diseases, such as hypertension, atherosclerosis and restenosis after injury. AT(1) receptor (AngII type-1 receptor), a G-protein-coupled receptor, mediates most of the physiological and pathophysiological actions of AngII, and this receptor is predominantly expressed in cardiovascular cells, such as VSMCs (vascular smooth muscle cells). AngII activates various signalling molecules, including G-protein-derived second messengers, protein kinases and small G-proteins (Ras, Rho, Rac etc), through the AT(1) receptor leading to vascular remodelling. Growth factor receptors, such as EGFR (epidermal growth factor receptor), have been demonstrated to be 'trans'-activated by the AT(1) receptor in VSMCs to mediate growth and migration. Rho and its effector Rho-kinase/ROCK are also implicated in the pathological cellular actions of AngII in VSMCs. Less is known about the endothelial AngII signalling; however, recent studies suggest the endothelial AngII signalling positively, as well as negatively, regulates the NO (nitric oxide) signalling pathway and, thereby, modulates endothelial dysfunction. Moreover, selective AT(1)-receptor-interacting proteins have recently been identified that potentially regulate AngII signal transduction and their pathogenic functions in the target organs. In this review, we focus our discussion on the recent findings and concepts that suggest the existence of the above-mentioned novel signalling mechanisms whereby AngII mediates the formation of cardiovascular diseases.

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. CONCLUSIONS: 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.

Activation of endothelial nitric oxide synthase by the angiotensin II type 1 receptor.

Enhanced angiotensin II (AngII) action has been implicated in endothelial dysfunction that is characterized as decreased nitric oxide availability. Although endothelial cells have been reported to express AngII type 1 (AT1) receptors, the exact role of AT1 in regulating endothelial NO synthase (eNOS) activity remains unclear. We investigated the possible regulation of eNOS through AT1 in bovine aortic endothelial cells (BAECs) and its functional significance in rat aortic vascular smooth muscle cells (VSMCs). In BAECs infected with adenovirus encoding AT1 and in VSMCs infected with adenovirus encoding eNOS, AngII rapidly stimulated phosphorylation of eNOS at Ser1179. This was accompanied with increased cGMP production. These effects were blocked by an AT1 antagonist. The cGMP production was abolished by a NOS inhibitor as well. To explore the importance of eNOS phosphorylation, VSMCs were also infected with adenovirus encoding S1179A-eNOS. AngII did not stimulate cGMP production in VSMCs expressing S1179A. However, S1179A was able to enhance basal NO production as confirmed with cGMP production and enhanced vasodilator-stimulated phosphoprotein phosphorylation. Interestingly, S1179A prevented the hypertrophic response similar to wild type in VSMCs. From these data, we conclude that the AngII/AT1 system positively couples to eNOS via Ser1179 phosphorylation in ECs and VSMCs if eNOS and AT1 coexist. However, basal level NO production may be sufficient for prevention of AngII-induced hypertrophy by eNOS expression. These data demonstrate a novel molecular mechanism of eNOS regulation and function and thus provide useful information for eNOS gene therapy under endothelial dysfunction.

Current understanding of the mechanism and role of ROS in angiotensin II signal transduction.

Reactive oxygen species (ROS) are proposed to induce cardiovascular diseases, such as atherosclerosis and hypertension, through several mechanisms. One such mechanism involves ROS acting as intracellular second messengers, which lead to induction of unique signal transductions. Angiotensin II (AngII), a potent cardiovascular pathogen, stimulates ROS production through vascular NADPH oxidases. The ROS production induced by AngII activates downstream ROS-sensitive kinases that are critical in mediating cardiovascular remodeling. Recent advances in gene transfer/knockout techniques have lead to numerous in vitro and in vivo studies that identify the potential components and mechanisms of ROS signal transduction by AngII which promote cardiovascular remodeling. In this review, we will focus our discussion on the signal transduction research elucidating ROS production and function induced by AngII using currently available molecular biotechnologies.

p21-activated kinase 1 participates in vascular remodeling in vitro and in vivo.

Vascular smooth muscle cell hypertrophy, proliferation, or migration occurs in hypertension, atherosclerosis, and restenosis after angioplasty, leading to pathophysiological vascular remodeling. Angiotensin II and platelet-derived growth factor are well-known participants of vascular remodeling and activate a myriad of downstream protein kinases, including p21-activated protein kinase (PAK1). PAK1, an effector kinase of small GTPases, phosphorylates several substrates to regulate cytoskeletal reorganization. However, the exact role of PAK1 activation in vascular remodeling remains to be elucidated. Here, we have hypothesized that PAK1 is a critical target of intervention for the prevention of vascular remodeling. Adenoviral expression of dominant-negative PAK1 inhibited angiotensin II-stimulated vascular smooth muscle cell migration. It also inhibited vascular smooth muscle cell proliferation induced by platelet-derived growth factor. PAK1 was activated in neointima of the carotid artery after balloon injury in the rat. Moreover, marked inhibition of the neointima hyperplasia was observed in a dominant-negative PAK1 adenovirus-treated carotid artery after the balloon injury. Taken together, these results suggest that PAK1 is involved in both angiotensin II and platelet-derived growth factor-mediated vascular smooth muscle cell remodeling, and inactivation of PAK1 in vivo could be effective in preventing pathophysiological vascular remodeling.

Novel role of protein kinase C-delta Tyr 311 phosphorylation in vascular smooth muscle cell hypertrophy by angiotensin II.

We have shown previously that activation of protein kinase C-delta (PKC delta) is required for angiotensin II (Ang II)-induced migration of vascular smooth muscle cells (VSMCs). Here, we have hypothesized that PKC delta phosphorylation at Tyr(311) plays a critical role in VSMC hypertrophy induced by Ang II. Immunoblotting was used to monitor PKC delta phosphorylation at Tyr(311), and cell size and protein measurements were used to detect hypertrophy in VSMCs. PKC delta was rapidly (0.5 to 10.0 minutes) phosphorylated at Tyr(311) 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 delta wild-type compared with VSMCs expressing control vector, whereas the enhancements were markedly diminished in VSMCs expressing a PKC delta Y311F mutant. Also, these responses were significantly inhibited in VSMCs expressing kinase-inactive PKC delta K376A compared with VSMCs expressing control vector. From these data, we conclude that not only PKC delta kinase activation but also the Src-dependent Tyr(311) 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.