ANG II stimulates phospholipase D through PKCζ activation in VSMC: implications in adhesion, spreading, and hypertrophy

Abstract

lates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-(PKC) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKC and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKC activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKC activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKC decreased ANG II-induced PLD activity. Moreover, depletion of PKC with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKC in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKC and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKC and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKC and suggest a crucial role of PKC-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension. vascular smooth muscle cells; protein kinase C; angiotensin II PHOSPHOLIPASE D (PLD) catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and choline. Activation of PLD by neurotransmitters, hormones, or growth factors has been implicated in a wide range of biological actions, including cellular trafficking, inflammatory and immune response, mitogenesis, cellular differentiation, and apoptosis (23). To date, two PLD isoforms, PLD1 and PLD2, have been cloned and characterized (7, 11). PLD1 is activated by direct binding of classical PKCs and small G proteins of Arf and Rho family (13,26). PLD2 requires phosphatidylinositol 4,5-bisphosphate and is not or is less responsive to Arf, Rho, and protein kinase C (PKC) than PLD1 (4, 19).ANG II contributes to the regulation of vascular tone and blood pressure (5). Hypertrophy of vascular smooth muscle cells (VSMC) induced by ANG II is an important feature of hypertension, and the structural changes in the vessel wall contribute to the increase in vascular resistance (25). VSMC response to ANG II, i.e., hyperplasia vs. hypertrophy, is determined by autocrine expression of transforming growth factor-␤ (TGF-␤) (12). ANG II in most cases does not increase DNA synthesis in VSMC. However, it increases protein synthesis and causes hypertrophy of confluent quiescent VSMC (3). A key event in neointima formation and atherogenesis is the migration of VSMC and fibroblasts into the intima. This is controlled by cytokines and extracellular matrix components within the microenvironment of the dis...

Heterogeneity of Phospholipase D Activation by Angiotensin II, Lysophosphatidylcholine, and Insulin in Human Endothelial Cells

“…This result significantly extends our knowledge regarding the effects of AII in the human vasculature-previous research has been limited to the demonstration of AII-mediated PLD activation in vascular smooth muscle cells (VSMCs) in animal models (Touyz and Schiffrin 2001;Andreson et al 2004;Parmentier et al 2006). A role for the endothelium in AII-induced vasoconstriction is supported by previous data, indicating an impaired vasoconstrictor effect of AII in endothelial denuded arteries in the rat (Chen et al1995).…”

ANGIOTENSIN II INCREASES TISSUE‐SPECIFIC INHIBITOR OF METALLOPROTEINASE‐2 EXPRESSION IN RAT AORTIC SMOOTH MUSCLE CELLS IN VIVO: EVIDENCE OF A PRESSURE‐INDEPENDENT EFFECT

“…Angiotensin (Ang) II has a wide spectrum of biological effects on VSMC, acting on cell proliferation, inflammation, apoptosis, extracellular matrix protein synthesis and degradation 10–14 …”

Cytochrome P450 1B1 Is Critical for Neointimal Growth in Wire‐Injured Carotid Artery of Male Mice

“…DNA synthesis for proliferation was determined by measuring the incorporation of [ 3 H]‐thymidine in VSMCs, as described 16. Subconfluent VSMCs from Cyp1b1 +/+ and Cyp1b1 −/− mice were treated with PDGF‐BB (20 ng/mL) or its vehicle in the presence or absence of Tempol (2 mmol/L) in 24‐well plates along with addition of [ 3 H]‐thymidine (0.5 μCi/well), and radioactivity in the cells was measured after 24 hours.…”