R W Alexander

Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells.

The signaling pathways involved in the long-term metabolic effects of angiotensin II (Ang II) in vascular smooth muscle cells are incompletely understood but include the generation of molecules likely to affect oxidase activity. We examined the ability of Ang II to stimulate superoxide anion formation and investigated the identity of the oxidases responsible for its production. Treatment of vascular smooth muscle cells with Ang II for 4 to 6 hours caused a 2.7 +/- 0.4-fold increase in intracellular superoxide anion formation as detected by lucigenin assay. This superoxide appeared to result from activation of both the NADPH and NADH oxidases. NADPH oxidase activity increased from 3.23 +/- 0.61 to 11.80 +/- 1.72 nmol O2-/min per milligram protein after 4 hours of Ang II, whereas NADH oxidase activity increased from 16.76 +/- 2.13 to 45.00 +/- 4.57 nmol O2-/min per milligram protein. The NADPH oxidase activity was stimulated by exogenous phosphatidic and arachidonic acids and was partially inhibited by the specific inhibitor diphenylene iodinium. NADH oxidase activity was increased by arachidonic and linoleic acids, was insensitive to exogenous phosphatidic acid, and was inhibited by high concentrations of quinacrine. Both of these oxidases appear to reside in the plasma membrane, on the basis of migration of the activity after cellular fractionation and their apparent insensitivity to the mitochondrial poison KCN. These observations suggest that Ang II specifically activates enzyme systems that promote superoxide generation and raise the possibility that these pathways function as second messengers for long-term responses, such as hypertrophy or hyperplasia.

Hypertension and the Pathogenesis of Atherosclerosis Oxidative Stress and the Mediation of Arterial Inflammatory Response: A New Perspective

Hypertension is a risk factor for the development of atherosclerosis, although the mechanisms have not been well elucidated. As the cellular and molecular mechanisms of the pathogenesis of atherosclerosis and the effects of hypertension are being more clearly defined, it becomes apparent that the two processes have certain common mechanisms. The endothelium is a likely central focus for the effect of both diseases. There is increasing evidence that atherosclerosis should be viewed fundamentally as an inflammatory disease. Atherogenic stimuli such as hyperlipidemia appear to active the inflammatory response by causing expression of mononuclear leukocyte recruiting mechanisms. The gene for one of these, the vascular cell adhesion molecule-1, is controlled at least in part by transcriptional factors regulated by oxidative stress, which modifies the redox state of the endothelial cell. Alterations in the redox state of the arterial wall also may contribute to vascular smooth muscle cell growth. In a somewhat parallel fashion, there is evidence that hypertension may also exert oxidative stress on the arterial wall. This article reviews evidence that leads to the postulate that hypertension predisposes to and accelerates atherosclerosis at least in part because of synergy between elevated blood pressure and other atherogenic stimuli to induce oxidative stress on the arterial wall.Abstract Hypertension is a risk factor for the development of atherosclerosis, although the mechanisms have not been well elucidated. As the cellular and molecular mechanisms of the pathogenesis of atherosclerosis and the effects of hypertension are being more clearly defined, it becomes apparent that the two processes have certain common mechanisms. The endothelium is a likely central focus for the effect of both diseases. There is increasing evidence that atherosclerosis should be viewed fundamentally as an inflammatory disease. Atherogenic stimuli such as hyperlipidemia appear to activate the inflammatory response by causing expression of mononuclear leukocyte recruiting mechanisms. The gene for one of these, the vascular cell adhesion molecule-1, is controlled at least in part by transcriptional factors regulated by oxidative stress, which modifies the redox state of the endothelial cell. Alterations in the redox state of the arterial wall also may contribute to vascular smooth muscle cell growth. In a somewhat parallel fashion, there is evidence that hypertension may also exert oxidative stress on the arterial wall. This article reviews evidence that leads to the postulate that hypertension predisposes to and accelerates atherosclerosis at least in part because of synergy between elevated blood pressure and other atherogenic stimuli to induce oxidative stress on the arterial wall.

Angiotensin II Signaling in Vascular Smooth Muscle: New Concepts

Angiotensin II is a multifunctional hormone that affects both contraction and growth of vascular smooth muscle cells through a complex series of intracellular signaling events initiated by the interaction of angiotensin II with the AT1 receptor. The cellular response to angiotensin II is multiphasic, involving stimulation within seconds of phospholipase C and Ca2+ mobilization; activation within minutes of phospholipase D, A2, protein kinase C, and MAP kinase; and stimulation after a period of hours of gene transcription and NADH/NADPH oxidase activity. Angiotensin II also activates numerous intracellular tyrosine kinases. In this respect, it shares some aspects of signaling with growth factor and cytokine receptors, including activation of phospholipase C-gamma, src, and ras; association of shc with grb2; and stimulation of the Jak/STAT pathway. The cellular events responsible for this unique series of events may involve receptor movement and the creation of a signaling domain. Elucidation of these pathways is important to our understanding of AT1 receptor function as a final effector of the renin-angiotensin system.

Molecular biology of the renin-angiotensin system

T he renin-angiotensin system is one of the major regulators of blood pressure and fluid and electrolyte homeostasis. Its primary components are 1) angiotensinogen, a large globular protein that serves as the substrate for 2) renin, the enzyme that catalyzes the proteolytic conversion of angiotensinogen to the decapeptide angiotensin I; 3) angiotensin converting enzyme, a dipeptidyl carboxypeptidase that converts angiotensin I to the octapeptide angiotensin II; 4) angiotensin II itself; and 5) the angiotensin II receptor, responsible for transducing the cellular effects of angiotensin II (Figure 1). Binding of the final product of this enzymatic cascade (angiotensin II) to its receptor mediates vasoconstriction and aldosterone and catecholamine release, as well as drinking, secretion of prolactin and adrenocorticotrophic hormone, and glycogenolysis. Historically, this hormonal system has been viewed as a systemic one, the various components of which were derived from different organs and were delivered to their site of action by the circulatory system. More recent evidence using molecular and biochemical approaches to angiotensin physiology raises the possibility that there are distinct, local renin-angiotensin systems with different mechanisms of regulation. Local reninangiotensin systems have been proposed in the vasculature,2 brain,3 heart,4 and kidney,5 but incontrovertible evidence for the existence of all components of the system in physiologically relevant amounts and relationships remains elusive. Although the role of these putative local systems is unknown, it is interesting to speculate that they may serve as a mechanism for limiting the actions of angiotensin II to a specific organ system or physiological event. Molecular biological and sophisticated biochemical measurements have opened a new era in our understanding of this important hormonal system. Not only have these techniques brought to the forefront the controversy surrounding the anatomic location of the individual components, but they also have provided insight into the regulation of the expression and formation of each protein or peptide and opened new strategies for therapeutic manipulation. The recent cloning of the AT, receptor6,7 and the development of pharmacological agents that distinguish this receptor from the closely related, but structurally distinct, AT2 binding site provide a fertile and relatively unexplored field for

Angiotensin II Type 1 Receptor: Relationship With Caveolae and Caveolin After Initial Agonist Stimulation

Caveolae are membrane domains that have been implicated in signal transduction, and caveolins are major structural components of these domains. We found that all reported caveolin isoforms (caveolin-1, -2, and -3) were expressed in vascular smooth muscle cells (VSMCs); however, only caveolin-1 mRNA was regulated by angiotensin II (Ang II). Ang II (100 nmol/L) increased caveolin-1 mRNA, with a peak at 2 hours (193+/-6% of control, P<0.01, n=4). In contrast, Ang II significantly decreased caveolin-1 protein, with a nadir at 4 hours (64+/-5% of control, P<0.01, n=6). [35S]Methionine labeling showed that Ang II increased caveolin biosynthesis (226+/-33% of control labeling at 4 hours), suggesting that the transient decrease in caveolin protein levels is due to increased degradation. When cells were fractionated with sucrose, on agonist stimulation, AT1 receptors appeared in fraction 5 where caveolin was fractionated. This migration was blocked by low temperature and treatment with phenylarsine oxide, interventions that interfere with agonist-induced Ang II type 1 (AT1) receptor sequestration and tonic phase signaling. In addition, caveolin-1 coimmunoprecipitates with AT1 receptor only on agonist stimulation. These data support the concept that the caveola is a specialized signaling domain in VSMCs that can be dynamically accessed by the AT1 receptor. Because of the signaling and coupling proteins that are localized in caveolae and because of evidence that these proteins may interact directly with caveolin, caveola-AT1 receptor interaction likely represents an important focus for dynamic control of receptor signaling in VSMCs.

Linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, stimulate c-Fos, c-Jun, and c-Myc mRNA expression, mitogen-activated protein kinase activation, and growth in rat aortic smooth muscle cells.

Previous studies from other laboratories suggest that linoleic acid and its metabolites, hydroperoxyoctadecadienoic acids, play an important role in modulating the growth of some cells. A correlation has been demonstrated between hydroperoxyoctadecadienoic acids and conditions characterized by abnormal cell growth such as atherosclerosis and psoriasis. To determine if linoleic acid and its metabolites modulate cell growth in atherosclerosis, we measured DNA synthesis, protooncogene mRNA expression, and mitogen-activated protein kinase (MAPK) activation in vascular smooth muscle cells (VSMC). Linoleic acid induces DNA synthesis, c-fos, c-jun, and c-myc mRNA expression and MAPK activation in VSMC. Furthermore, nordihydroguaiaretic acid, a potent inhibitor of the lipoxygenase system, significantly reduced the growth-response effects of linoleic acid in VSMC, suggesting that conversion of linoleic acid to hydroperoxyoctadecadienoic acids (HPODEs) is required for these effects. HPODEs also caused significant induction of DNA synthesis, protooncogene mRNA expression, and MAPK activation in growth-arrested VSMC, suggesting that linoleic acid and its metabolic products, HPODEs, are potential mitogens in VSMC, and that conditions such as oxidative stress and lipid peroxidation which provoke the production of these substances may alter VSMC growth.

Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells.

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that cytosolic phospholipase A2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase.

Molecular structure and transcriptional function of the rat vascular AT1a angiotensin receptor gene.

Rat vascular angiotensin receptors (AT1a receptors) are encoded by two mRNA transcripts sharing an identical receptor coding sequence but differing in their 5 and 3 untranslated sequences. We screened male Sprague-Dawley rat genomic libraries to clone the vascular AT1a receptor gene. Two sets of overlapping clones were isolated that encode over 90 kb of genomic sequence around the AT1a receptor gene. Four overlapping clones were identified from the 5 flanking portion of the gene. These contain the promoter region and two exons, 141 bp and 89 bp in size, respectively, encoding the alternatively spliced 5 untranslated mRNA sequence. Six additional clones overlap each other but do not overlap the set of clones from the 5 flanking region of the gene. These contain a single 1977-bp exon that encodes 900 bp of the 5 and 3 untranslated sequences in addition to a 1077-bp open reading frame identical to that found in vascular smooth muscle cell AT1a receptor cDNAs. Primer extension and RNase protection studies indicate that the transcription start site for this gene begins 9 bp upstream from the most 5 sequence found within the AT1a receptor cDNAs. Our mapping studies of the cloned gene, which so far includes an uncloned gap within the second intron, indicate that the transcription start site is no less than 67 kb upstream from the receptor coding exon. Promoter-reporter assays were performed by transfection of vascular smooth muscle cells with deletions of a 3.2-kb promoter region fused to a luciferase cDNA reporter plasmid. Relatively strong basal transcriptional activity is observed from the 5-most 2 kb of the promoter and diminishes markedly with deletions within 1 kb of the early promoter region, suggesting strong promoter elements in the more upstream regions of the gene. Deletion of a 53-bp early promoter region containing the transcription start site and a putative TATA box completely abolishes the ability of upstream elements to drive transcription of the luciferase cDNA. These results indicate that we have isolated the AT1a receptor gene and its functional promoter.

Abdominal coarctation increases insulin-like growth factor I mRNA levels in rat aorta.

We have previously demonstrated specific insulin-like growth factor I (IGF I) mRNA transcripts in cultured endothelial and vascular smooth muscle cells and postulated an important role for IGF I in blood vessel growth responses. The purpose of this study was to characterize IGF I gene expression in a model of aortic coarctation hypertension in the rat. This high-renin model of hypertension is associated with hyperplastic vascular responses. Northern analysis of rat aorta demonstrated four specific IGF I mRNA transcripts sized 7.6, 4.6, 1.8, and 0.9-1.2 kb. Quantitation of aortic IGF I mRNA levels by solution hybridization/RNase protection assay demonstrated induction of IGF I transcripts in the hypertensive aorta; levels more than doubled at 7 days and were still significantly elevated 21 days after coarctation. In situ hybridization analysis indicated that IGF I transcripts were localized primarily to adventitial surfaces in normotensive aorta, with minimal signal detected over vascular cells. In hypertensive aortas, there was an increase in IGF I transcripts primarily over vascular smooth muscle cells. Thus, vascular IGF I gene expression is induced in this model of high-renin hypertension. IGF I may play an important role in autocrine/paracrine-mediated vessel wall remodeling in hypertension.

Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.

Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.