Differential mechanisms of angiotensin II and PDGF-BB on migration and proliferation of coronary artery smooth muscle cells. Journal of Molecular and Cellular Cardiology, 45 (2). pp. 198-208.

Angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) are associated with excessive cell migration, proliferation and many growth-related diseases. However, whether these agents utilise similar mechanisms to trigger vascular pathologies remain to be explored. The effects of Ang II and PDGF-BB on coronary artery smooth muscle cell (CASMC) migration and proliferation were investigated via Dunn chemotaxis assay and the measurement of [ 3 H]thymidine incorporation rates, respectively. Both atherogens produced similar degrees of cell migration which were dramatically inhibited by mevastatin (10 nM). However, the inhibitory effects of losartan (10 nM) and MnTBAP (a free radical scavenger; 50  M) were found to be unique to Ang II-mediated chemotaxis. In contrast, MnTBAP, apocynin (an antioxidant and phagocytic NADPH oxidase inhibitor; 500 µM), mevastatin and pravastatin (100 nM) equally suppressed both Ang II and PDGF-BB-induced cellular growth. Although atherogens produced similar changes in NADPH oxidase, NOS and superoxide dismutase activities, they differentially regulated antioxidant glutathione peroxidase activity which was diminished by Ang II and unaffected by PDGF-BB. Studies with signal transduction pathway inhibitors revealed the involvement of multiple pathways i.e. protein kinase C, tyrosine kinase and MAPK in Ang II- and/or PDGF-BB-induced aforementioned enzyme activity changes. In conclusion, Ang II and PDGF-BB may induce coronary atherosclerotic disease formation by stimulating CASMC migration and proliferation through agent-specific regulation of oxidative status and utilisation of different signal transduction inhibited Ang II- but not PDGF-BB-dependent chemotaxis. These findings imply


Introduction
Coronary atherosclerosis, associated with enhanced oxidative stress, continues to be the leading cause of morbidity and mortality in the Western World. Atherosclerosis is a progressive inflammatory disease that ultimately leads to formation of advanced or complicated focal lesions which develop subsequent to a series of specific cellular and molecular responses including enhanced vascular smooth muscle cell (VSMC) proliferation and migration [1]. Several agents including platelet-derived growth factor (PDGF) exacerbate atherogenesis through inducing VSMC proliferation and plaque neovascularisation. The HMG-CoA reductase inhibitors (statins) e.g. simvastatin have been implicated in the suppression of PDGF-induced DNA synthesis in human glomerular mesangial cells in addition to reducing the risk of primary and secondary events [2][3][4]. Another non-lipidlowering effect of statins is their ability to prevent migration of VSMC to PDGF by blocking the production of isoprenoids which are required for prenylation of small GTP-binding proteins, such as Ras and Rho, involved in cell proliferation and migration control [5]. Hence, the suppression of PDGF-induced mitogenesis and migration should dramatically limit its atherogenic effects. Similar to PDGF, angiotensin II (Ang II), the active component of the renin-angiotensin system, stimulates VSMC proliferation and migration and is therefore implicated in atherogenesis [6]. Previous data have concluded that Ang II type 1 receptor (AT 1 R) blockers (ARB) exert direct anti-atherosclerotic effects [7,8]. Ang II displays its proatherogenic effects through AT 1 R stimulation and consequent NADPH oxidase enzyme activation to generate excess levels of superoxide anion (O 2 .-), a reactive oxygen species (ROS) [9]. The effects of ROS range from eliciting vasoconstriction to scavenging nitric oxide (NO), an endogenous vasodilator produced from L-arginine by NO synthase (NOS) [10,11]. Under physiological conditions, O 2 .is converted to H 2 O 2 by superoxide dismutases (SOD) which is then further metabolised to H 2 O by catalase and glutathione peroxidase (GPx) [12]. However, in pathological conditions, O 2 .may lead to atherosclerotic plaque formation via diminished concentrations and/or aberrant regulations of antioxidant enzymes.
In addition to their respective roles in SMC proliferation, migration and oxidative stress, Ang II and PDGF share somewhat similar signal transduction characteristics such as activation of tyrosine kinases and mitogen-activated protein kinases (MAPK) [13,14].
Furthermore, recent data have implicated PDGF-BB in Ang II-induced chemotaxis and revealed the ability of Ang II to activate PDGF- receptor [15,16]. Taken these close functional interactions into account, the present study examined whether mechanistic dis/similarities exist between these atherogens to trigger vascular pathologies. To this end the putative differences in: i-oxidative status as assessed by pro-and anti-oxidant enzyme activities; ii-proliferative and migratory responses; iii-signal transduction pathways involved in atherogen-mediated oxidative stress regulation, and iv-the correlation of ARBs, an antioxidant, a free radical scavenger or statins to cell growth and enzyme activities were investigated using coronary artery smooth muscle cells (CASMC) exposed to physiopathological concentrations of Ang II or PDGF-BB.

Human CASMC culture and characterisation
Human CASMC (n=3 donors) were purchased from Cambrex (UK) and cultured in SmGM-2 according to manufacturer's instructions. Cells grown on coverslips were rinsed with ice-cold PBS and fixed in 4% formaldehyde for 20 min at room temperature before permeabilisation with 0.1% Triton X-100. To characterise the cells as SMC they were stained with a monoclonal antibody raised against SM -actin for 1 h in the dark at 4 o C prior to examination by confocal microscopy (Micro Radiance, Bio-Rad, UK). Moreover, the cells were stained positive for SM myosin, calponin and caldesmin by the commercial company.
CASMC between passages 4 and 8 were used in the current study.

Manipulation of cell growth
To exclude potential effects produced by differences in cell density, an identical number of cells was seeded into flasks (0.5 x 10 6 ). Cell passages were performed using a 1:3 ratio and culture medium was replaced every 24 h. This ensured that the number of mitoses was unaltered amongst experiments.

NOS Assay
NOS activity was determined, in cell homogenates, using the NOSdetect assay kit (Alexis Biochemicals were expressed as pmol L-citrulline/mg protein/min. In parallel experiments, the cellular homogenates were incubated with N  -hydroxy-nor-Larginine (NOHA; 100 M) for 30 min to inactivate arginase activity before measuring the NOS activities.

Nitrite Detection
Nitrite levels were measured by Griess reaction as an index of NO generation following conversion of nitrate to nitrite by nitrate dehydrogenase [19]. An aliquot of the cellular homogenate was mixed with an equal volume of Griess reagent (sulfanilamide 1% w/v, naphthylethylenediamine dihydrochloride 0.1% w/v and orthophosphoric acid 2.5% v/v) and incubated at room temperature for 10 min prior to measurement of absorbances at 540 nm.
The amount of nitrite formed was compared to those of known concentrations of sodium nitrite and normalised to the protein content of the respective flask.

GPx assay
The GPx activity was measured using a specific assay kit (Merck Biosciences) based on the reduction of oxidised glutathione, produced by GPx-mediated reduction of hydroperoxide, by glutathione reductase and NADPH during which the oxidation of NADPH to NADP + was accompanied by a decrease in absorbance at 340 nm. Briefly, 100 µl of assay buffer and 50 µl of co-substrate mixture were added to 20 µl of untreated and treated CASMC homogenate, in triplicate, in a 96-well ELISA plate. Reactions were initiated by the addition of 20 µl cumene hydroperoxide and the sample absorbances were read every 60 sec for 5 min at 340 nm. The reaction rate per min was determined for the blank reaction and then subtracted from the reaction rates for the analysed samples. GPx activity was then calculated using the extinction coefficient for NADPH at 340 nm (0.00373 µM -1 ). One unit of GPx was defined as the activity that converts 1 mM of reduced glutathione per litre per min at 25 o C.

SOD Assay
The SOD assay kit (Merck Biosciences) utilising a tetrazolium salt for detection of O

Evaluation of cell viability
To detect cytotoxicity of ROS generating enzyme inhibitors, CASMC were incubated with the aforementioned compounds for 75 min. A small aliquot was then incubated with 0.1% trypan blue for 4 min and viewed under a light microscope. By counting 100 cells, the percentage of viable cells was calculated.

Statistical Analysis
To test for directed migration (chemotaxis) the Rayleigh test for unimodal clustering of directions was applied to the data and p<0.01 was chosen as the criterion for rejecting the null hypothesis of random directionality. Where there was significant unimodal clustering the mean direction and its 95% confidence interval were calculated and Tukey's post hoc analysis was conducted. Results for proliferation and enzyme activity assays are presented as meanSEM. Statistical analyses were performed by two-way analysis of variance (ANOVA) followed by Bonferroni-Dunn's post hoc analysis and p<0.05 was considered significant.

Effects of Ang II and PDGF-BB on CASMC migration
Ang II ( PDGF-BB (5-50 ng/ml) also significantly enhanced CASMC chemotaxis. However, unlike Ang II, the PDGF-evoked responses were not affected by MnTBAP and losartan but markedly inhibited by mevastatin (Fig. 1B).
The cells used in chemotaxis experiments were serum starved for 18 h to exclude the migratory effects of serum in order to detect the specific and directed migration produced by a given atherogen. As the cells in the absence of stimuli such as serum or atherogens generated aberrant undirected migratory responses, they could not be used as positive controls.
However, the impact of a given treatment on atherogen-mediated chemotaxis was assessed using the cells exposed to Ang II or PDGF-BB alone as controls.

Effects of Ang II and PDGF-BB on CASMC proliferation
Ang II (5-50 nM) produced significant increases in cell numbers in a dose-dependent manner as assessed by cell counting (0.88 ± 0.07 x 10 6 vs.  Fig. 2A-B). Treatment of cells with a phagocytic inhibitor of NADPH oxidase i.e. apocynin (100 M) also produced substantial decreases in cell growth ( Fig. 2A-B). However, the specific inhibitors of xanthine oxidase (allopurinol, 10-100 M), cyclooxygenase (indomethacin, 5-50M) and mitochondrial complex I inhibitor (rotenone, 5-50 M) did not alter cellular growth rate (data not shown).

Effects of Ang II and PDGF-BB on enzyme activities
Overnight incubation of CASMC with the higher dose of Ang II (50 nM) and both concentrations of PDGF-BB (5 and 50 ng/ml) dramatically decreased NOS activity.
Ang II and PDGF-BB increased NADPH oxidase activity in a dose-dependent fashion which was significantly diminished by mevastatin, losartan and apocynin (Fig. 4A-B). The level of changes observed in NADPH oxidase activities measured using the total cellular homogenates were shown to be representative of those obtained with membrane fractions (Table 1). To assess the potential toxic effects of ROS-generating enzyme inhibitors that were used in NADPH oxidase assays to eradicate the contributions of enzymes, other than NADPH oxidase, to overall O 2 .formation, CASMC viability rates were measured which revealed no significant difference amongst treatment groups ( Table 2).
The levels of mitochondrial (MnSOD) and cytosolic SOD (CuZn-SOD) activities were also elevated with both atherogens where treatments with mevastatin, losartan and apocynin alone abolished both SOD activities and significantly decreased them when used in combination with either atherogen (Table 3).
Atherogenic agents differentially regulated GPx activity that was dose-dependently decreased by Ang II and unaffected by PDGF-BB. It was shown that, when used alone, mevastatin but not losartan or apocynin significantly decreased basal GPx activity. Although these agents significantly decreased GPx activity in the presence of PDGF-BB, only mevastatin was shown to be suppressive to Ang II (Fig. 5A-B).

Effects of Ang II and PDGF-BB on O 2 .and nitrite levels
The differences observed in CASMC NADPH oxidase and NOS activity following overnight exposure to Ang II (5-50 nM) or PDGF-BB (5-50 ng/ml) were mimicked by the differences observed in the levels of their end products; O 2 .and nitrite, an indirect marker used for estimation of NO production, respectively (Table 4).

Effects of inhibition of signal transduction pathways on enzyme activities
Suppression of tyrosine kinase (TLCK; 50 μM), MAPK (PD98059; 10 μM) or protein kinase C (Bis-I; 5 μM) using the indicated specific inhibitors led to atherogenand enzymespecific changes. Individual inhibition of tyrosine kinase and MAPK diminished both PDGF-BB-and Ang II-mediated GPx activity. However, co-incubation of these inhibitors with Ang II enhanced GPx activities compared to control cells and high dose Ang II-treated cells.
Significant increase in GPx activity was also observed in PDGF-BB and TLCK co-incubated versus control cells (Fig. 6A-B). The inhibition of each pathway reduced NADPH oxidase and CuZn-SOD enzyme activities compared to higher dose PDGF and Ang II-treated cells by almost 50% while concomitantly increasing NOS activity in these cells (Tables 5 and 6).

Discussion
The major conclusions to be drawn from this study are that Ang II and PDGF-BB distinctly regulate CASMC proliferation, migration and oxidative status through involvement of multiple enzyme systems and signal transduction pathways. These findings provide crucial evidence on the mechanisms whereby these agents may contribute to vasculopathologies in addition to their well-established vasoconstrictor and mitogenic effects.  [24]. The critical roles of Ras and other small GTP-binding proteins including p21 Rac in the assembly of the NADPH oxidase enzyme system, cell proliferation, NOS activity and thus NO generation may help explain the beneficial effects of statins on preventing and/or regressing atherosclerotic plaque development [25,26].
In addition to cell migration, proliferation which contributes to in-stent restenosis is also considered a key feature of atherosclerosis [27]. Hence, the present study investigated the correlation between atherogenic agents and CASMC proliferation and revealed that both atherogens increased O 2 .production and CASMC proliferation in a dose-dependent manner which were suppressed by MnTBAP, apocynin and mevastatin. Taken with the migration studies these data imply that while ROS modulate both Ang II-mediated CASMC proliferation and migration, they are solely associated with PDGF-induced proliferative processes. It is of note that differential regulation of VSMC proliferation and chemotaxis is not unique to the agents used in this study, as similar effects have been generated with VAS2870, a novel NADPH oxidase inhibitor, in rat thoracic aorta SMC [29]. In this context, a previous study has attributed normal coronary endothelial cell growth to NADPH oxidase-derived O 2 .bioavailability, a finding supported in this study by inability of other ROSgenerating enzymes to alter CASMC proliferation rates [26].

NO inhibits proliferation of several cells and thus prevents atherosclerotic disease
progression [28]. Since, similar to previous studies, no significant differences in iNOS activity were detected in CASMC incubated with/out Ang II and PDGF-BB, the enhanced cell growth was investigated in relation to potential differences in eNOS activity and nitrite levels, a surrogate marker of NO [30,31]. These studies revealed that both Ang II and PDGF-BB  [12]. This study has shown that both Ang II and PDGF-BB have increased both CuZn-SOD (the main isotype which accounts for ~80% of total SOD activity) and MnSOD activities, a strong indicator of elevated intracellular oxidative stress, which were significantly attenuated by mevastatin, apocynin or losartan. Considering the suppressive effects of these agents on NADPH oxidase and hence O 2 .production, these results were somewhat expected. However, the data pertaining to GPx activity were rather unexpected and displayed an atherogen-dependent regulation in that while Ang II elicited a dose-dependent decrease in its activity, PDGF-BB did not affect its basal activity. Besides, while mevastatin, losartan and apocynin significantly decreased GPx activity when co-incubated with PDGF-BB, both losartan and apocynin failed to alter Ang II-induced GPx activity.
Variety of signal transduction pathways including tyrosine kinases, protein kinase C and MAPK activated by Ang II and PDGF are implicated in VSMC differentiation, migration and proliferation [35,36]. In this study, selective inhibition of these signal transduction pathways to examine the putative links between their impaired regulation and promotion of pathological cascades have shown an atherogen-and/or enzyme-specific changes in enzyme activities. For example, suppressions of tyrosine kinase and MAPK enhanced Ang II-but not PDGFmediated GPx activity. In contrast, selective inhibition of each pathway displayed similar effects on NADPH oxidase, CuZn-SOD and NOS activities. The similarities in these responses can in part be explained by the well-documented presence of a cross-talk between G-protein coupled AT 1 R and the PDGF receptor tyrosine kinase in vascular SMCs and thus the ability of Ang II to elicit responses unique to growth factor stimulation [15,16,37].
In conclusion, statins, free radical scavengers and ARB may suppress the early events in atherogenesis by markedly inhibiting overall oxidative stress status, SMC migration and/or proliferation. Intracellular antioxidants and ARB suppress Ang II-mediated but not PDGF-BB-induced chemotaxis despite equally blocking cellular proliferation to both agents.
Considering the involvement of different signal transduction pathways in the regulation of pro-and anti-oxidant enzyme activities, it is important to reiterate that Ang II and PDGF-BB       Losartan 1μM Apocynin 500μM      34 Table 5. The effects of Ang II on NAD(P)H oxidase, CuZn-SOD and NOS activities in the absence or presence of a serine protease inhibitor tosyl-L-lysine chloromethyl ketone (TLCK), a MAPK inhibitor (PD98059) and a protein kinase C inhibitor (Bis-I).

PDGF-BB
The enzyme activities were measured in coronary artery smooth muscle cells (CASMC) cultured with Ang II for 24 hours in the absence or presence of indicated signal NOS (pmol L-citrulline/mg protein/min) Table 6. The effects of PDGF-BB on NAD(P)H oxidase, CuZn-SOD and NOS activities in the absence or presence of a serine protease inhibitor tosyl-L-lysine chloromethyl ketone (TLCK), a MAPK inhibitor (PD98059) and a protein kinase C inhibitor (Bis-I).
The enzyme activities were measured in coronary artery smooth muscle cells (CASMC) cultured with PDGF-BB for 24 hours in the absence or presence of indicated signal transduction pathway inhibitors. Results expressed as mean ± SEM from three separate experiments. NOS: nitric oxide synthase; SOD: superoxide dismutase. *p<0.05 difference compared to basal untreated group. † p<0.05 difference compared to PDGF-BB 50ng/ml treatment group.