Skq1 Synthesis Essay

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Hypoxia-inducible factor-1 (HIF-1) is a key hypoxia-inducible transcription factor responsible for the adaptation of cells and tissues to low oxygen by regulating such responses as cell metabolism, proliferation and survival, erythropoiesis, and angiogenesis (Semenza, 2003). HIF-1 binds to hypoxia-response elements (HRE) found in promoter or enhancer DNA regions of target hypoxia-inducible genes that include vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), nitric oxide synthases, and likely between 100 and 200 others (Kaelin and Ratcliffe, 2008).

The active HIF-1 complex is a heterodimer consisting of an oxygen-sensitive HIF-1α subunit and a constitutively expressed HIF-1β subunit. HIF-1α possesses an oxygen-dependent degradation domain (ODDD) containing two key proline residues which are, in the presence of oxygen, hydroxylated by HIF prolyl-hydroxylases (PHDs; Kaelin and Ratcliffe, 2008). HIF-1α prolyl-hydroxylation allows for recognition by pVHL, the product of the von Hippel-Lindau tumor suppressor gene, the substrate recognition component of an E3 ubiquitin ligase complex that polyubiquitinates and targets HIF-1α for proteasomal degradation. In hypoxic conditions, low oxygen leads to HIF-1α stabilization due to inhibition of prolyl-hydroxylation and subsequent decreases in HIF-1α ubiquitination and degradation (Cockman et al., 2000; Epstein et al., 2001; Schofield and Ratcliffe, 2004).

In addition to hypoxia, there are also different nonhypoxic HIF activators that include growth factors, hormones, cytokines, and viral proteins (Dery et al., 2005). In vascular smooth muscle cells (VSMCs) we have shown that angiotensin II (Ang II), a vasoactive hormone linked to many cardiovascular functions and diseases, is a potent HIF-1 activator (Richard et al., 2000). Treatment of VSMCs with Ang II regulates HIF-1 by common as well as distinct mechanisms from hypoxia. Ang II increases HIF-1α stability (as in hypoxia), but additionally increases HIF-1α translation and transcription (Page et al., 2002, 2008; Lauzier et al., 2007).

Reactive oxygen species (ROS) are small, extremely reactive molecules due to their unpaired valence electrons. High ROS concentrations are very damaging to cells, because they lead to a free-radical–mediated chain reaction–targeting proteins, lipids, polysaccharides, and DNA (Turrens, 2003). However, low and intermediary levels of ROS are crucial to a number of cellular signaling events (Cai et al., 2002; Varela et al., 2004; Felty et al., 2005; Kimura et al., 2005b; Rhee, 2006; Zhang et al., 2007; Lassegue and Griendling, 2010). There are a number of intracellular ROS generators. Two of the most documented include the NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase and the mitochondria. The NADPH oxidase is a complex comprised of membrane bound (Nox1-4 and p22phox) and cytoplasmic (p47phox, p67phox and Rac) subunits (Babior, 2004). Once activated, cytoplasmic subunits interact with their membrane-bound counterparts and create an active complex which oxidizes NADPH, leading to the generation of ROS. In VSMCs, a large body of literature indicates that NADPH oxidase–derived ROS (noxROS) are a primary source of ROS after Ang II treatment (Sorescu et al., 2001; Garrido and Griendling, 2009). On the other hand, mitochondria mainly produce ROS at complex I and complex III of the electron transport chain, whereas a total of nine other ROS-producing sites have been identified (Andreyev et al., 2005). Interestingly, complex III produces ROS in both the mitochondrial matrix and the intermembrane space, whereas the other sites produce superoxide solely in the mitochondrial matrix (Zhang et al., 2007). Mitochondrial-derived ROS (mtROS) have recently gained attention in vascular biology and were shown to be increased by Ang II (Kimura et al., 2005a; Doughan et al., 2008; Nozoe et al., 2008).

Our studies have shown that the generation of ROS during Ang II treatment is essential for both increased HIF-1α translation and stability. Ang II augments HIF-1α translation by noxROS-dependent increases in receptor tyrosine kinase transactivation and phosphatidylinositol 3-kinase (PI3K)/p70S6 kinase (p70S6K) pathway activation (Page et al., 2002; Lauzier et al., 2007). Additionally, our recent work indicated that ROS/H2O2 increased HIF-1α protein accumulation through a Fenton reaction where Fe2+, an essential PHD cofactor, is oxidized to Fe3+, leading to the inactivation of PHD, decreased HIF-1α hydroxylation, and increased HIF-1α protein stabilization (Page et al., 2008).

Here, we set out to identify the ROS generator responsible for HIF-1 stabilization during Ang II treatment. Surprisingly, we show that targeting noxROS generation did not play a major role in HIF-1α stabilization and accumulation in VSMCs treated with Ang II. Instead, the specific targeting of mtROS generation by inhibition of mitochondrial complex III and by using mitochondrial-targeted antioxidants completely abolishes HIF-1α stabilization and accumulation during the treatment of VSMCs with Ang II by reestablishing PHD activity and HIF-1α hydroxylation. Additionally, Ang II–stimulated HIF-1 activity in VSMCs, measured by HIF-1–mediated target gene expression and VSMC migration depends on mtROS generation. These results indicate that mtROS are essential intermediates for HIF-1 induction and activation by Ang II in VSMCs.

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Ang II, PDGF-BB, myxothiazol, and stigmatellin were all from Sigma-Aldrich (St. Louis, MO). MG132 was from EMD Chemicals (Gibbstown, NJ). VAS2870 was from Vasopharm (Wuerzburg, Germany). Anti-HIF-1α antibody was raised in our laboratory in rabbits immunized against the last 20 amino acids of the C terminal of human HIF-1α (Richard et al., 1999). Anti-hydroxylated HIF-1α antibodies, against either hydroxylated Pro402 or hydroxylated Pro564 of human HIF-1α, were obtained as previously described (Chan et al., 2002, 2005). Monoclonal anti-phospho-p42/p44 MAPK and anti-α-tubulin antibodies were from Sigma-Aldrich. Anti-phospho-p70S6 kinase (Thr389), p70S6 kinase, phospho-AKT, AKT, and monoclonal p38 MAP kinase antibodies were from Cell Signaling (Beverly, MA). p22phox and Rieske Fe-S protein monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p38 (pTpY180/182) polyclonal antibody was from Invitrogen (Carlsbad, CA). Total polyclonal p42/p44 MAPK antibody was from Upstate/Millipore (Billerica, MA). Anti-glutathione S-transferase (GST) antibody was from Novus Biologicals (Littleton, CO). Monoclonal HA.11 antibody was from Covance (Emeryville, CA). Horseradish peroxidase-coupled anti-mouse and anti-rabbit antibodies were from Promega (Madison, WI). A GST-HIF-1α fusion protein, comprised of amino acids 344-582 from human HIF-1α, and pVHL-hemagglutinin (HA) constructs were kind gifts from Drs. Jacques Pouysségur and Peter Ratcliffe respectively. The mitochondrial antioxidant, SkQ1 was from Drs. Vladimir Skulachev and Oleg Fedorkin at the Institute of Mitoengineering, Moscow State University (Skulachev et al., 2009).

Cell Culture

Rat VSMCs were isolated from the thoracic aortas of 6 wk-old male Wistar rats by enzymatic dissociation (Owens et al., 1986). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin, 50 U/ml streptomycin (Invitrogen, Carlsbad, CA) and 2 mM glutamine in a humid atmosphere (5% CO2, 95% air). Cells were serially passaged once reaching confluence, and only cells between passages 4 and 12 were used for experiments. In all experiments, cells were deprived of serum overnight. For MitoSOX assays, cells were cultured in phenol red–free DMEM. Hypoxic conditions were obtained by placing cells in a sealed hypoxic workstation (Ruskinn Technology, Bridgend, United Kingdom). The oxygen level in this workstation was maintained at 1%, with the residual gas mixture containing 94% nitrogen and 5% CO2.

Western Blot Analysis

Confluent cells were lysed in a 2× Laemmli buffer. Protein concentration was determined by Lowry assay. Samples were resolved on SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (PVDF, Immobilon-P, Millipore). Proteins were analyzed using indicated antibodies and visualized with an enhanced chemiluminescence (ECL) system (GE Healthcare, Piscataway, NJ) or with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE). Western blots were quantified using Odyssey quantification software (LI-COR Biosciences) or ImageJ (National Institutes of Health;

RNA Silencing

Cells were seeded in six-well plates at a density of 6 × 105 cells/well. Twenty-four hours after plating, small interfering RNA (siRNA) oligonucleotides were transfected by CaPO4 precipitation. Thirty-six hours after transfection, cells were then deprived of FBS for 16 h before stimulation. All siRNAs were obtained from Applied Biosystems/Invitrogen and the specific sequences used are as follows: rat HIF-1α (accession no. NM_024359): sense: 5′-AGGACAAGUCACCACAGGAuu-3′; rat p22phox (accession no. NM_024160): sense: 5′-AAGUACCUGACCGCUGUGGtg-3′; rat Rieske Fe-S (accession no. NM_001008888; predesigned siRNA (accession no. s145479): sense 5′-GCAUGAUUUAGAGCGUGUAtt-3′. As a control oligonucleotide, Silencer Negative Control 2 siRNA was used.

mtROS Assay

mtROS production was determined using MitoSOX Red mitochondrial superoxide indicator (Invitrogen), which is selectively targeted to the mitochondria and is fluorescent upon ROS oxidation. MitoSOX was used according to the manufacturer's protocol and published literature (Robinson et al., 2008). Briefly, VSMCs were seeded on glass-bottomed cell culture dishes and serum-deprived overnight in phenol red-free DMEM. VSMCs were then incubated with MitoSOX (1 μM) for a total of 1 h before analysis. Cells were then pretreated or not with inhibitors as indicated 20 min after MitoSOX addition. Ang II (100 nM) was added during the final 20 min. Cell imaging was performed with a FV1000 confocal microscope equipped with a live cell apparatus (60× oil, 1.4 NA) driven by FluoView software (Olympus, Tokyo, Japan). Fluorescence quantification was performed using the Measure Integrated Density function of the ImageJ software.

HIF-1α Half-Life Analysis

VSMCs were pretreated with stigmatellin or myxothiazol for 20 min and then treated with Ang II. After 4 h, cycloheximide (30 μg/ml) was added for different periods of time (up to 30 min). VSMCs were then lysed in a 2× Laemmli buffer. HIF-1α and α-tubulin protein levels were evaluated by Western blotting followed by quantification and the ratio of HIF-1α to α-tubulin was determined. HIF-1α half-life under experimental conditions was estimated by plotting data as the HIF-1–α-tubulin ratio versus time under cycloheximide treatment.

pVHL Capture Assay

pVHL capture assay was performed as previously described (Page et al., 2008). Briefly, VSMCs were grown to confluence, serum-deprived for 16 h, and stimulated as indicated for 4 h. Cells were washed once in PBS and twice in ice-cold HEB buffer (20 mM Tris, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol). Cells were then lysed using a Dounce homogenizer, and cytoplasmic extracts were isolated by centrifugation (20,000 × g). Cytoplasmic extracts (250 μg) were incubated with Sepharose-bound GST-HIF-1α (30 μg) for 1 h at room temperature. The Sepharose-bound GST-HIF-1α was then washed with NETN buffer (150 mM NaCl, 0.5 mM EDTA, 20 mM Tris, pH 8.0, 0.5% Igepal, 100 μM deferoxamine) and incubated overnight with in vitro–translated pVHL-HA in NETN at 4°C. Samples were then washed with NETN, denatured with 2× Laemmli buffer, resolved in SDS-polyacrylamide gels (12%), and revealed using anti-HA and anti-GST antibodies.

Intracellular Ascorbate Assay

VSMCs were grown to confluence on 100-mm plates in culture media supplemented with ascorbate (250 μM). Cells were serum-deprived for 16 h in ascorbate supplemented DMEM, and fresh DMEM without ascorbate was added 1 h before treatments. Cells were washed with PBS and lysed in a 90% methanol/1 mM EDTA solution. Samples were briefly sonicated and centrifuged at 20,000 × g. Ascorbate levels were then analyzed by spectrophotometry using a modified protocol (Queval and Noctor, 2007). Samples were diluted in 0.2 mM NaH2PO4, pH 5.6, and absorbance was measured at 265 nm. Ascorbate peroxidase (0.4 U) was added to samples for 5 min, and the absorbance was again measured at 265 nm. Ascorbate concentrations were determined as the difference in absorbance prior and after the addition of ascorbate peroxidase. A Lowry protein assay was used for normalization of the samples.

Northern Blot Analysis

Confluent VSMCs were lysed with TRIzol reagent (Invitrogen). RNA and protein were extracted according to the manufacturer's protocol. RNA concentration was then quantified using RiboGreen (Invitrogen). RNA (20 μg) was resolved on 1% agarose/6% formaldehyde gels and transferred to Hybond N+ nylon membranes (GE Healthcare) before hybridization with a radioactive cDNA probes comprising either the total coding sequence of the mouse VEGF gene and the human GLUT1 gene or the 900-base pair coding sequence of the human HIF-1α gene. An oligonucleotide probe against 18S rRNA was used as a loading control. Northern blots were quantified using a STORM phosphoimaging system equipped with ImageQuant software (GE Healthcare Life Sciences).

Cell Migration Assay

VSMCs were serum deprived for 16 h before determination of cell migration by Boyden chamber assay using a 48-well reusable chamber (Neuroprobe, Gaithersburg, MD). Polycarbonate PVPF membranes (8.0 μm; GE Water & Process Technologies, Trevose, PA), coated with rat tail type I collagen (VWR International, West Chester, PA), were used to separate the upper and lower chambers. VSMCs were added to the upper chamber at a density of 5 × 104 cells per well in DMEM containing 0.1% fatty-acid-free BSA. VSMCs were allowed to migrate toward the lower chamber containing media with or without 100 nM Ang II and inhibitors for 4 h at 37°C. After treatment, membranes were fixed with methanol and subjected to Giemsa staining (Thermo Fisher Scientific, Waltham, MA). Removal of nonmigrated cells was performed by wiping membranes with cotton swabs. Membranes were then air dried on a microscope slide. Cells were counted in three different fields. Results shown are means ± SD of four independent experiments performed in triplicate.

Statistical Analysis

For Western blot and imaging studies, results are representative of three independent experiments. Unless otherwise noted, quantification results are expressed as means ± SEM of three independent experiments. Statistical analyses of different experiments were performed using InStat 3 (GraphPad). Unless otherwise noted, Student's t tests were performed. Results were deemed significant if they attained a 95% confidence level (p < 0.05).

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noxROS Do Not Play a Major Role in HIF-1α Accumulation by Ang II in VSMCs

A large body of evidence indicates that the NADPH oxidase is the principle ROS generator in Ang II–treated VSMCs (Lyle and Griendling, 2006; Garrido and Griendling, 2009). We therefore decided to first examine whether NADPH oxidase–derived ROS were implicated in HIF-1α accumulation under nonhypoxic conditions. VAS2870, a specific NADPH oxidase inhibitor, was first used to inhibit noxROS generation (ten Freyhaus et al., 2006). As seen in Figure 1A, when used at reported functional concentrations (10–50 μM), VAS2870 showed no effect on increased HIF-1α protein levels in VSMCs treated with Ang II. We also investigated the role of noxROS using a molecular approach. Inhibiting the p22phox subunit can abolish noxROS generation because this membrane bound protein is an essential member of the NADPH oxidase complex (Ushio-Fukai et al., 1996; Ambasta et al., 2004; Hanna et al., 2004; Modlinger et al., 2006). Targeting the p22phox subunit with a specific siRNA was able to efficiently decrease p22phox protein levels in VSMCs (Figure 1B). However, p22phox silencing was unable to modify HIF-1α protein levels after a treatment with Ang II. Activation of the PI3K pathway has been linked to NADPH oxidase activity and noxROS generation (Page et al., 2002; Baumer et al., 2008). We therefore determined if NADPH oxidase inhibition with VAS2870 or p22phox siRNA inhibited the PI3K pathway by evaluating p70S6 kinase (p70S6K) phosphorylation, a downstream target of this pathway. As seen in Figure 1, A and B, Ang II–induced p70S6K phosphorylation was indeed inhibited by pretreatment of VSMCs with both VAS2870 and p22phox siRNA. These results indicate that noxROS generation does not play a major role in regulating HIF-1α accumulation in VSMCs during Ang II stimulation.

Figure 1.

noxROS do not regulate HIF-1α accumulation. (A) Quiescent VSMCs were pretreated with VAS2870 (indicated concentrations) for 20 min and then treated or not with Ang II (100 nM) for 4 h. HIF-1α, phospho-p70S6K, total-p70S6K, and α-tubulin levels were then analyzed by Western blot. (B) VSMCs were transfected with a siRNA against p22phox or a control sequence. Quiescent VSMCs were treated or not with Ang II (100 nM) for 4 h. HIF-1α, p22phox, phospho-p70S6K, total-p70S6K, and α-tubulin levels were then analyzed by Western blot.

mtROS Are Essential for Increased HIF-1α Accumulation by Ang II in VSMCs

We next attempted to determine the role of mitochondrial-generated ROS in HIF-1α accumulation by Ang II. We first used a pharmacological approach, using stigmatellin and myxothiazol, two inhibitors of the mitochondrial electron transport chain (ETC), specifically complex III. Interestingly, both compounds strikingly inhibited HIF-1α accumulation by Ang II (Figure 2A). However, myxothiazol and stigmatellin were unable to inhibit HIF-1α accumulation by CoCl2 (Supplemental Figure 1), a HIF-1 inducer that potently inhibits HIF-1α hydroxylation and functions independently of oxygen levels and mtROS generation (Sanjuan-Pla et al., 2005). Using MitoSOX, a mitochondrial-targeted probe for detecting ROS, results show that treatment of VSMCs with Ang II does indeed increase mtROS generation (Figure 2B). As expected, stigmatellin and myxothiazol both inhibited mtROS generation in VSMCs by Ang II (Figure 2B), whereas VAS2870 was ineffective (Supplemental Figure 2A). Our previous studies have shown that diphenyleneiodonium (DPI), which blocks ROS generation through its activity as a flavoprotein-containing enzyme inhibitor, potently represses Ang II–induced HIF-1α accumulation in VSMCs (Richard et al., 2000; Page et al., 2002). In accordance with our past studies, DPI also strikingly decreased mtROS generation (Supplemental Figure 2B). Finally, our results show that pretreatment of VSMCs with stigmatellin or myxothiazol does not affect other Ang II/AT1-related cellular signaling because p70S6K, p38MAPK, AKT, and p42/44 MAPK activation/phosphorylation levels were not modified (Supplemental Figure 3).

Figure 2.

mtROS regulate HIF-1α accumulation. (A) Quiescent VSMCs were pretreated with stigmatellin (Stig, 1 μM) or myxothiazol (Myx, 1 μM) for 20 min and then treated or not with Ang II (100 nM) for 4 h. HIF-1α and α-tubulin levels were analyzed by Western blot. (B) VSMCs were seeded on 35-mm glass-bottomed cell culture dishes and loaded with MitoSOX (1 μM) for 1 h. During the final 40 min, cells were pretreated or not with stigmatellin (1 μM) or myxothiazol (1 μM). Ang II (100 nM) was then added 20 min before imaging as described in Materials and Methods. Differential interference contrast microscopy (DIC) was used for whole cell imaging. Scale bars, 10 μm. Bottom panel is the quantification of results from B. Data are expressed as arbitrary units of MitoSOX fluorescence and are an average ± SD of five independent experiments, n > 50 cells. ***p < 0.001 compared with untreated VSMCs. †††p < 0.001 compared with Ang II–treated VSMCs.

Two additional approaches were used to confirm the role played by mtROS in HIF-1α induction in VSMCs after Ang II treatment. First, we investigated the importance of the Rieske Fe-S protein, an essential member of complex III. Without Rieske Fe-S, mtROS are not generated because complex III cannot initiate the Q cycle (Brunelle et al., 2005). As seen with complex III inhibitors, targeting Rieske Fe-S with a specific siRNA led to an inhibition of HIF-1α accumulation after Ang II treatment (Figure 3A). Second, we utilized a mitochondrial-targeted antioxidant: SkQ1. Developed to investigate the role of mitochondrial ROS in ageing, this Skulachev (Sk) ion is strongly localized to the mitochondria. SkQ1 is a “rechargeable” antioxidant because it is reduced by the mitochondrial ETC (Skulachev et al., 2009). As seen in Figure 4A, the pretreatment of VSMCs with SkQ1 before Ang II treatment blocked HIF-1α protein accumulation. As expected, both the siRNA knockdown of Rieske Fe-S and the pretreatment of VSMCs with SkQ1 abolished mtROS generation by Ang II as determined by MitoSOX staining (Figures 3B and 4B and Supplemental Figure 4). Taken together, our results show that in VSMCs, mtROS generation is essential for Ang II–induced HIF-1α accumulation.

Figure 3.

mtROS regulate HIF-1α accumulation. (A) VSMCs were transfected with a siRNA against Rieske Fe-S protein (100 nM) or a control sequence. Cells were then serum-deprived overnight and treated or not with Ang II (100 nM) for 4 h. HIF-1α, Rieske Fe-S protein, and α-tubulin levels were analyzed by Western blot. (B) VSMCs seeded on glass-bottomed cell culture dishes were transfected and treated as in A. Cellular mtROS levels were analyzed by MitoSOX staining as described in Figure 2. Scale bars, 10 μm.

Figure 4.

mtROS regulate HIF-1α accumulation. (A) Quiescent VSMCs were pretreated with SkQ1 (500 nM) and then treated or not with Ang II (100 nM) for 4 h. HIF-1α, phospho p42/p44 MAPK, total p42/p44 MAPK and α-tubulin levels were analyzed by Western blot. (B) VSMCs seeded on glass-bottomed cell culture dishes were transfected and treated as in A. Cellular mtROS levels were analyzed by MitoSOX staining as described in Figure 2. Scale bars, 10 μm.

mtROS Are Involved in Increasing HIF-1α Stabilization

Our previous work demonstrated that ROS were essential in HIF stabilization during the treatment of VSMCs with Ang II (Page et al., 2008). We therefore attempted to determine the importance of mtROS on HIF-1α stabilization. We first examined the role of mtROS on HIF-1α half-life using the general protein synthesis inhibitor, cycloheximide (Laughner et al., 2001; Page et al., 2008; Michaud et al., 2009). Incubating cells with cycloheximide after HIF-1 induction blocks de novo protein synthesis. Thus, cellular HIF-1α protein levels are dependent on the maintenance of protein stabilization mechanisms. In these experimental conditions, changes in HIF-1α half-life would therefore reflect changes in HIF-1α stability. Here, HIF-1α half-life was determined by Western blotting and the quantification of HIF-1α levels over time in the presence of cycloheximide. When VSMCs were treated with Ang II, HIF-1α half-life was 20 ± 2.8 min (Figure 5). However, when VSMCs were pretreated with either stigmatellin or myxothiazol, HIF-1α half-life under Ang II treatment was significantly decreased (7.0 ± 0.5 and 5.5 ± 0.6 min, respectively). It is important to note that the inhibition of mtROS generation did not modify increased HIF-1α mRNA levels by Ang II (Supplemental Figure 5). This last result was expected because our studies have shown that Ang II–increased HIF-1α mRNA levels are ROS-independent in VSMCs (Page et al., 2002). Therefore, our results indicate that HIF-1α stability is increased through Ang II–dependent mtROS generation in VMSC.

Figure 5.

mtROS regulate HIF-1α half-life. (A) Quiescent VSMCs were pretreated with stigmatellin (1 μM) or myxothiazol (1 μM) for 20 min and then treated or not with Ang II (100 nM) for 4 h. HIF-1α half-life was measured by then treating cells with cycloheximide (30 μg/ml) for the indicated times to block all de novo protein synthesis. HIF-1α and α-tubulin levels were then analyzed by Western blot. (B) HIF-1α protein levels in A were quantified using the Odyssey Infrared Imaging System and normalized to α-tubulin. HIF-1α half-life was determined by plotting data as HIF-1α–α-tubulin ratio versus time under cycloheximide treatment. Data expressed are an average ± SEM of three independent experiments. **p < 0.01 compared with Ang II–treated VSMCs.

We then investigated whether mtROS could act on two main steps involved in HIF-1α protein degradation; HIF-1α prolyl hydroxylation and HIF-1α binding to pVHL. To analyze HIF-1α hydroxylation status, specific antibodies against hydroxylated HIF-1α on its two key ODDD proline residues were used (Chan et al., 2002; Chan et al., 2005). As seen in Figure 6, VSMCs treated with Ang II show decreased HIF-1α hydroxylation, most notably on the Pro402 residue (Page et al., 2008). More interestingly, the decrease in HIF-1α hydroxylation by Ang II was reversed when VSMCs were pretreated with either stigmatellin or myxothiazol. Additionally, VAS2870 had no significant effect on HIF-1α's hydroxylation status during Ang II treatment. A crucial event leading to HIF-1α protein degradation is its binding to the product of the von Hippel-Lindau tumor suppressor gene, pVHL. Binding to pVHL is a direct result of HIF-1α hydroxylation (Ivan et al., 2001). To determine the effect of Ang II–induced mtROS generation on pVHL binding, a pVHL capture assay was used. A GST-HIF-1α fusion protein, comprised of amino acids 344-582 from human HIF-1α, was subjected to modification by Ang II–treated VSMCs cell extracts followed by interaction with in vitro–translated pVHL. As we previously demonstrated, Ang II treatment led to decreased HIF-1α binding to pVHL (Figure 7). More interestingly, pretreatment of VSMC with stigmatellin or myxothiazol was able to restore HIF-1α binding to pVHL (Figure 7A). Finally, VAS2870 or siRNA against p22phox had no significant effect on HIF-1α's hydroxylation status or levels of pVHL binding during Ang II treatment (Figures 6 and 7). It is important to note that inhibitors or p22phox had no significant effect on HIF-1α hydroxylation or pVHL binding in cells not treated with Ang II (data not shown). Taken together, these results indicate that Ang II–generated mtROS are essential for HIF-1α prolyl-hydroxylase inactivation in VSMCs.

Figure 6.

mtROS regulate HIF-1α hydroxylation. Quiescent VSMCs were pretreated with stigmatellin (1 μM), myxothiazol (1 μM), or VAS2870 (10 μM) for 20 min. Cells were then treated with MG132 (20 μM) to increase HIF-1 levels and stimulated with Ang II (100 nM) or CoCl2 (200 μM) for 4 h. Nuclear extracts (100 μg) were analyzed by Western blot with anti-hydroxylated-Pro402-HIF-1α (P402-OH), anti-hydroxylated-Pro564-HIF-1α (P564-OH), anti-HIF-1α and anti-α-tubulin antibodies (top). Western blots in A were quantified with ImageJ or the Odyssey Infrared Imaging System using α-tubulin as a loading control (bottom panel). Results are expressed as a percentage of hydroxylated HIF-1 (normalized to total HIF-1α protein levels) on Pro402 (■) or Pro564 (▩) compared with untreated cells and are an average ± SEM of three independent experiments. **p < 0.01 and *p < 0.05 compared with untreated VSMCs.

Figure 7.

mtROS inhibit VHL-HIF-1α binding. (A) Quiescent VSMCs were pretreated with stigmatellin (1 μM), myxothiazol (1 μM), or VAS2870 (10 μM) for 20 min and subsequently treated or not with Ang II (100 nM) or CoCl2 for 4 h. Cytoplasmic extracts were incubated with GST-HIF-1α coupled to Sepharose beads for 1 h. Samples were then incubated in the presence of in vitro–translated pVHL and resolved by SDS-PAGE (12%). Immunoblotting was performed using anti-HA (pVHL) and anti-GST antibodies (A, top). Western blots were quantified with the Odyssey Infrared Imaging System using GST-HIF-1α as a loading control (A, bottom panel). Results are expressed as a percentage of pVHL binding normalized to GST-HIF-1α protein levels compared with untreated cells and are an average ± SEM of four independent experiments. **p < 0.01 and *p < 0.05 compared with untreated VSMCs. (B) VSMCs were transfected with a siRNA against p22phox or a control sequence. Quiescent VSMCs were treated or not with Ang II (100 nM) for 4 h. VHL-capture assay was then performed as in A.

Our previous studies showed that HIF-1α prolyl hydroxylase activity is inactivated during Ang II stimulation by a ROS-induced depletion of intracellular ascorbate (Page et al., 2008). To determine whether mtROS were implicated in depleting intracellular ascorbate, ascorbate levels were measured in Ang II–stimulated VSMCs that were pretreated with stigmatellin or myxothiazol. As seen in Supplemental Figure 6, Ang II treatment decreased intracellular ascorbate levels by 65.2 ± 6.2%. The inhibition of mtROS generation with stigmatellin or myxothiazol was able to completely reverse intracellular ascorbate depletion caused by the treatment of VSMCs with Ang II. These results indicate that mtROS generation by Ang II in VSMCs leads to reduced cellular ascorbate concentrations, decreased HIF-1α hydroxylation, decreased HIF-1α binding to pVHL and increased HIF-1α stability.

mtROS Are Required for Increased HIF-1 Transcriptional Activity by Ang II

The importance of mtROS for HIF-1 transcriptional activity was analyzed by evaluating the expression of two HIF-1 target genes: VEGF and Glut-1. As seen in Figure 8, Ang II increased the expression of both VEGF and Glut-1, an effect that was strongly inhibited by pretreating cells with either stigmatellin or myxothiazol. As with HIF-1α protein accumulation, a maximal inhibition was observed with 1 μM of either complex III inhibitor (Supplemental Figure 7). To confirm that VEGF and Glut-1 expression during Ang II treatment were indeed dependent on HIF-1 in VSMCs, we targeted HIF-1α expression with a specific siRNA. Our results show that the expression of these two transcripts is nearly exclusively dependent on HIF-1 activation (Supplemental Figure 8A). Effective silencing of HIF-1α can be observed in Supplemental Figure 8B. Taken together, these results indicate that Ang II regulates HIF-1–dependent genes such as VEGF and Glut-1 through mtROS generation in VSMCs.

Figure 8.

mtROS are essential for HIF-1–dependent activity. (A) Quiescent VSMCs were pretreated with stigmatellin (1 μM) or myxothiazol (1 μM) for 20 min and then treated or not with Ang II (100 nM) for 4 h. Total RNA was extracted and resolved on formaldehyde/agarose gels. Northern blotting was performed using specific VEGF- and Glut-1–radiolabeled probes. An 18S RNA probe was used as a control for gel loading. Northern blots were quantified by phosphoimaging using total ribosomal 18S RNA as a loading control (Bottom panels). Data expressed are an average ± SEM of four independent experiments. ***p < 0.001 compared with untreated VSMCs. †p < 0.05 compared with Ang II–treated VSMCs. ‡Nonsignificant compared with Ang II–treated VSMCs.

HIF-1 and mtROS Are Essential for Ang II–induced VSMC Migration

VSMC migration occurs during a number of different vascular pathologies, especially when vascular remodeling is involved (Gerthoffer, 2007). Ang II is a potent vascular remodeler and activator of VSMC migration (Xi et al., 1999; Guo et al., 2009). Additionally, HIF-1 was shown to be important for VSMC migration in hypoxic conditions (Corley et al., 2005; Osada-Oka et al., 2008). We wanted to determine whether HIF-1 and mtROS were involved in Ang II–mediated VSMC migration. For this assay, we used a Boyden chamber to analyze the potential of VSMCs to migrate across a porous membrane toward Ang II. HIF-1's role in VSMC migration was determined by targeting HIF-1α expression with a specific siRNA (Supplemental Figure 8). As seen in the top panel of Figure 9, Ang II significantly increased the migration of VSMCs transfected with a control siRNA that was nearly completely blocked in VSMCs transfected with a HIF-1α siRNA. To determine the role of mtROS in Ang II–mediated VSMC migration, VSMCs were treated with stigmatellin and myxothiazol during Boyden chamber assay. Both compounds potently inhibited VSMC migration toward Ang II (Figure 9, bottom panel). Interestingly, the effects of HIF-1 and mtROS on VSMC migration were specific to Ang II because VSMC migration toward PDGF-BB was unaffected by HIF-1α knockdown or complex III inhibition. Taken together, our results demonstrate the essential role of HIF-1 and mitochondrial ROS in Ang II–induced VSMC migration.

Figure 9.

mtROS are essential for Ang II–dependent VSMC migration. VSMCs were transfected with a siRNA against HIF-1 or a control sequence (top) or left untransfected (bottom). Cells were serum-starved 24 h after transfection. Quiescent VSMCs (5 × 105 cells/well) were placed in the upper level of a Boyden chamber and allowed to migrate through a collagen-coated microporous membrane for 4 h toward the lower chamber that contained 100 nM Ang II in DMEM only (top) or in DMEM supplemented or not with stigmatellin (1 μM) or myxothiazol (1 μM; bottom). Cells were then fixed, stained, and counted as described in Materials and Methods. Data expressed are an average ± SEM of three independent experiments performed in triplicate. ***p < 0.001 compared with untreated (−) VSMCs.

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Treating VSMCs with Ang II leads to altered HIF-1α proline hydroxylation, decreased pVHL binding, reduced HIF-1α ubiquitination, and proteasomal degradation leading to increased HIF-1α stabilization (Page et al., 2008). ROS generation plays a pivotal role in these Ang II–mediated effects on the HIF-1 system. Our current work now identifies mitochondrial-derived ROS as essential intermediates leading to the stabilization of HIF-1α, the activation of HIF-1, increased in HIF-1 target gene expression, and associated cellular effects, such as VSMC migration.

It is well accepted that the NADPH oxidase plays a primary role in VSMC biology. noxROS have been implicated in various VSMC signaling mechanisms originating from AT1 receptor activation after Ang II stimulation (Garrido and Griendling, 2009). noxROS have been implicated in increasing HIF-1α translation (Page et al., 2002; Lauzier et al., 2007). Additionally, a recent study showed the involvement of the NADPH oxidase in HIF-2 stabilization (Diebold et al., 2010). Because Ang II–induced HIF-1α stability was ROS dependent, our first studies pushed us to investigate the role of the NADPH oxidase and noxROS in HIF-1α stabilization. Under our conditions, the NADPH oxidase does not play a major role in HIF-1α accumulation. This is shown here by targeting p22phox by RNAi and using the specific NADPH inhibitor, VAS2870. However, siRNA against other NADPH oxidase subunits, including Nox1, Nox4 and p47phox, were equally ineffective to block HIF-1α accumulation after Ang II treatment. (D. A. Patten, E. L. Pagé, and D. E. Richard, unpublished observations). These unexpected results led us to demonstrate here that mitochondrial/complex III–generated mtROS are indispensable for HIF-1 activation by Ang II. It is important to note that the NADPH oxidase and the mitochondria are not the only ROS-generating systems in VSMCs (San Martin and Griendling, 2010). It is possible that the activity of other oxidases and ROS generators may affect HIF-1 activation at different levels. Continued studies in this area will clarify the role of other oxidases and ROS generators on HIF-1 activation in nonhypoxic conditions.

Our previous work described that DPI, which blocks ROS generation through its inhibitory effects on flavoprotein-containing enzymes, blocked HIF-1α accumulation during Ang II treatment. We suggested this was mediated through the inhibition of the NADPH oxidase. Not surprisingly, it has been reported that DPI is not a specific NADPH oxidase inhibitor (Hutchinson et al., 2007; Aldieri et al., 2008). Moreover, DPI inhibits all flavin-containing enzymes, including those found in the mitochondria (Li and Trush, 1998). Here, our work shows that DPI does indeed block mtROS generation in VSMCs during Ang II treatment and indicates that DPI blocks HIF-1α stabilization by Ang II.

Our results show that mtROS are essential for blocking HIF-1α hydroxylation and pVHL binding during Ang II treatment. It is well accepted that hydroxylation of proline residues 564 and 402 (Pro402 and Pro564) of human HIF-1α regulates pVHL-mediated proteasomal degradation (Ivan et al., 2001; Masson et al., 2001). Further studies indicated that the modification of only one of these proline residues is sufficient to stabilize HIF-1α (Chan et al., 2005). Our previous work demonstrated that treating VSMCs with Ang II primarily suppressed the hydroxylation of Pro402 (Page et al., 2008). In agreement with these observations, here we show that mtROS are responsible for decreasing Pro402 hydroxylation while Pro564 hydroxylation is mostly unaffected. Taken together, our results show that increased mtROS results in an inactivation of PHD.

H2O2 is known to be an important mediator of the effects of Ang II on VSMC (Zafari et al., 1998). Our previous studies demonstrated that H2O2 led to the inhibition of PHD activity, as measured by reduced pVHL/HIF-1α binding (Page et al., 2008). This indicates that superoxide, produced in the mitochondria, is transformed into H2O2, which effectively inhibits PHD activity. This inhibition most probably occurs due to the Fenton reaction and the oxidation of Fe2+ to Fe3+, which decreases available ascorbate, leading to decreased hydroxylation of HIF-1α on Pro402, impaired binding of pVHL to HIF-1α, and HIF-1α stabilization under normal oxygen conditions. These results also suggest a crucial role of superoxide dismutase (SOD) in HIF-1 activation by Ang II. Preliminary evidence suggest that SOD activity is indeed involved because diethylthiocarbamate (DETC), a SOD inhibitor, led to a significant inhibition of HIF-1α accumulation under Ang II treatment in VSMCs (G. A. Robitaille and D. E. Richard, unpublished observations). Further studies are needed to clearly identify the role of different SOD enzymes in HIF-1 activation. Given the importance of mtROS in HIF-1 induction, mitochondrial SOD2 may be of particular interest.

Because Ang II can activate both noxROS and mtROS in VSMCs after Ang II treatment, our results indicate a specificity of mtROS in regulating HIF-1α stabilization. The reason for this specificity for mtROS remains to be elucidated. Theoretically, mtROS differ from noxROS by only the area in which it is produced and the local expression of SOD. We hypothesize that local mtROS/H2O2 gradients exist around the mitochondria which is responsible for inhibition of PHD activity and HIF-1α stabilization signaling.

Exactly how Ang II increases mtROS remains to be elucidated. By using two ETC complex III inhibitors and a siRNA against the Rieske Fe-S, our results indicate an essential role of complex III. However, the exact sequence of events leading to complex III activation and mtROS generation after AT1 receptor activation remains unclear. In contrast to our findings, Kimura et al. (2005b) have proposed that in cardiac tissue, mtROS are produced during Ang II treatment through NADPH oxidase-derived ROS-induced ROS release (RIRR). In other words, the initial burst of noxROS leads to increased mtROS. Our results indicate that in VSMCs, RIRR does not occur because inhibition of the NADPH oxidase does not block Ang II–induced mtROS generation as measured by MitoSOX nor does it impede HIF-1 induction. It is important to note that results from Kimura et al. were based on data obtained using the classical NADPH oxidase inhibitor, apocynin. However, recent results now indicate that apocynin is not a simple NADPH oxidase inhibitor in vascular cells and can also act as an antioxidant (Heumuller et al., 2008). Additionally, it was shown that the expression of a dominant negative form of Rac1 can suppress mtROS generation by Ang II (Nozoe et al., 2008) Because Rac1 is required for proper NADPH oxidase assembly, this suggested that the NADPH oxidase is required for increasing mtROS (Griendling et al., 2000). Although the role of Rac1 on HIF-1 induction and activation has been described (Hirota and Semenza, 2001), Rac1 also regulates other cellular processes in VSMCs including cytoskeletal organization, gene transcription, cell proliferation and membrane trafficking through interactions with PI3K, p21-activated kinase (PAK), Ras, and p70 S6 kinase (Chou and Blenis, 1996; Kaibuchi et al., 1999; Liliental et al., 2000; Sun et al., 2000). Additionally, Rac1 has been shown to regulate HIF-1α mRNA expression through activation of the NF-κB pathway in different cell systems including VSMCs (Gorlach et al., 2003; Diebold et al., 2008; Kim et al., 2008). Finally, previous studies have shown that Rac1 does not affect mtROS generation in TNF-treated endothelial cells (Deshpande et al., 2000). Therefore, the effect of Rac1 on HIF-1 induction could potentially be attributed to any number of cellular processes not directly linked to mtROS generation. Finally, a recent study shows that inversely, mtROS can lead to NADPH oxidase activation (Lee et al., 2006). Given these divergent studies and our results, further investigation is needed to clearly delineate the mechanisms by which mtROS generation is activated in VSMCs after Ang II treatment.

HIF-1 regulation by Ang II occurs through three independent mechanisms. Until now, we have been unable to determine which mechanism played a primordial role in HIF-1 induction and activation. Our work here partly defines the relative contribution of the increased translation, transcription, and stability of HIF-1α by Ang II. We can now speculate that increased HIF-1α stability is of primary importance for HIF-1 regulation by Ang II. Also, because the inhibition of the NADPH oxidase had little or no effect on HIF-1α accumulation, and we have previously demonstrated that noxROS are important for increased HIF-1α translation by Ang II; our results indicate that increased HIF-1α protein translation is not essential for the prolonged accumulation of HIF-1α levels under Ang II treatment (Page et al., 2002). However, increased HIF-1α translation may be important for the rapid accumulation of HIF-1α after Ang II receptor activation.

Because mitochondrial oxidative damage is thought to contribute in a number of human degenerative diseases, the development of antioxidants that are targeted to the mitochondria has gained significant interest. Generally, mitochondrial antioxidants include an antioxidant moiety (ubiquinone, tocopherol, nitroxide) and a covalently attached lipophilic triphenylphosphonium cation that serves for specific uptake by the mitochondria (Smith et al., 2008). MitoQ and SkQ1 are two compounds that have been developed to specifically suppress mtROS generation. These two compounds have an added advantage over other mitochondrial-targeted antioxidants because they can be regenerated by accepting electrons from the respiratory chain. MitoQ has been shown to inhibit HIF-1α accumulation during hypoxia (Bell et al., 2007). However, recent evidence suggests that the concentration window for MitoQ's antioxidant properties is very small before displaying prooxidant properties (Antonenko et al., 2008). Using a different antioxidant moiety (plastoquinone), SkQ1 demonstrated more potent antioxidant properties and a larger functional concentration window than MitoQ (Skulachev et al., 2009). Here, we successfully used SkQ1 to decrease mtROS generation during Ang II treatment, which effectively inhibited HIF-1α accumulation. Our studies therefore confirm the effectiveness of mitochondrial antioxidants as inhibitors of HIF-1 activation under nonhypoxic conditions and point to interesting therapeutic leads. Our recent in vivo studies have demonstrated a potential role for nonhypoxic HIF-1 induction in vascular remodeling diseases (Lambert et al. 2010). Mitochondrial antioxidants, such as SkQ1, will prove interesting tools for further investigation in Ang II–mediated pathophysiological effects which involve VSMC migration, such as vascular remodeling.

Mitochondrial-derived ROS have gained substantial interest in the regulation of HIF-1 induction and activation. The hypoxic activation of HIF-1α has been shown by two independent groups to be regulated by mtROS (Chandel et al., 2000; Guzy et al., 2005). Additionally, a nonhypoxic activator of HIF-1, thrombopoietin (TPO), has been shown to control HIF-1α levels through the generation of mtROS (Yoshida et al., 2008). Although the NADPH oxidase has classically been shown as the primary ROS generator in VSMCs and noxROS to be involved in different signaling pathways, our study now identifies mtROS as an essential intermediate for PHD inactivation, HIF-1α stabilization, and HIF-1 activation when VSMCs are stimulated with Ang II. Taken together, these studies suggest that mitochondrial ROS are a common intermediate signal transducer between hypoxic and nonhypoxic stimuli leading to the activation of HIF-1.

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We thank Drs. Sébastien Bonnet, Marie-Claude Lauzier, Maude Michaud, and Elisabeth Pagé for their perspective and insightful advice. This work was supported by Grants MOP-49609 and MOP-102760 from the Canadian Institutes of Health Research (CIHR) and the Heart and Stroke Foundations of Québec and Canada. D.E.R. is the recipient of a CIHR New Investigator Award. D.A.P. held a Graduate Scholarship from La Société Québécoise d'Hypertension Artérielle (SQHA). V.E.R. holds a Banting and Best Canada Graduate Scholarship from the CIHR.

Abbreviations used:

Ang II
angiotensin II
electron transport chain
glucose transporter-1
hypoxia-inducible factor
hypoxia-response element
oxygen-dependent degradation domain
HIF prolyl-hydroxylase
von Hippel-Lindau tumor suppressor protein
reactive oxygen species
mitochondrial-derived ROS
NADPH oxidase–derived ROS
vascular endothelial growth factor
vascular smooth muscle cells.

This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (


The word “synthesis” is defined as a combination of elements to form a connected whole. Thus, a synthesis essay definition is an essay that combines different ideas into a whole to prove a point (otherwise called the thesis). Often, it comes with a text that you should analyze.

Table Of Contents

Writing Process

A key factor of writing a synthesis essay is an analysis of a given text or a prompt. In order to successfully analyze it, you must comprehend the text’s purpose, rhetoric, and the argument that the author’s claim, in other words, you are answering the question: “So what?”. Then, you must build your own claim, and write an essay around that.

Most Common Topics

A synthesis essay prompt must be negotiable. Like in the EssayPro's example above, Andrew Jackson’s negative views on Native American people were widely supported, today, however, they would be appalling. Depending on your assignment, you may have to choose a primary text. Choose a text that might have opposing viewpoints.

Good topics would be ones that are debatable, for example:

  • Daylight savings
  • Minimum wage
  • Abortion
  • Immigration policy
  • Global warming
  • Gun control
  • Social media

How Do I Write A Thesis?

Once you pick a topic of your paper, read your sources and establish your position. Make sure you thoroughly analyze the sources and get a good understanding of them, structure your claim or argument and write your thesis.

Example: Andrew Jackson’s fear of the Native American “savages” reflects the prejudices and ideas of the colonist people in the Union and the Congress.*

How Do I Write An Outline?

Creating an outline will help maintain the structure of your paper. If your essay is split into three parts, split your outline into three chunks. Paste supporting evidence, sub-arguments, and specific points in the appropriate sections. Make sure that every point somehow proves the claim in your thesis. Extra information or tangents will only hinder your essay. However, if information goes against your central claim, then you should acknowledge it as it will make your essay stronger. Make sure you have read all of your sources. When writing about the sources, do not summarize them; synthesis denotes analysis, not plot-summary.


  • Introduction
  • Thesis
    • Main point 1
    • Main point 2
    • Main point 3
  • Body
  • Main point 1
    • Evidence (quote from a source)
    • Analysis of Evidence
  • Main point 2
    • Evidence (quote from a source)
    • Analysis of Evidence
  • Main point 3
    • Evidence (quote from a source)
    • Analysis of Evidence
  • Conclusion
  • Restate main points and answer unanswered questions

Read more about how to write a great INTRODUCTION

How Do I Format My Essay?

The format depends on what style is required by your teacher or professor. The most common formats are: MLA, APA, and Chicago style. APA is used by fields of Education, Psychology, and Science. MLA is used for citing Humanities, and Chicago style is used for Business, History, and Fine Arts. Purdue Owl is a format guide that focuses mainly on MLA and APA, and Easybib is a citation multitool for any of your external sources.

Some key points are:

  • Times New Roman 12 pt font double spaced
  • 1” margins
  • Top right includes last name and page number on every page
  • Titles are centered
  • The header should include your name, your professor’s name, course number and the date (dd/mm/yy)
  • The last page includes a Works Cited

APA Format

Some key points are:

  • Times New Roman 12 pt font double spaced 1” margins
  • Include a page header on the top of every page
  • Insert page number on the right
  • An essay should be divided into four parts: Title Page, Abstract, Main Body, and References.

How do I write an AP English Synthesis Essay?

AP English Language and Composition is an extremely rigorous course that requires you to write essays that demonstrate deep understanding of the subject matter. In fact, if on the AP exam, your essay has perfect grammar and structure, you might still be awarded just 1 out of 9 points for not “defending, challenging, or qualifying your claim.” Sounds difficult, but it is doable. Before entering any AP class, it is best to read over the course overview and become familiar with the exam.

While writing, focus on the three branches of the AP English and Composition course: argument, synthesis, and rhetorical analysis.

Argument is the easiest component; create your claim and find specific supporting evidence. Convince your reader that you are right.

Synthesis requires you to read into multiple perspectives and identify an agreement and a disagreement between sources. This step is crucial to finding your own claim.

Rhetorical analysis deals with the author and his intentions. What was their purpose for writing this? Who is their intended audience? How does the author appeal to the audience and how does he structure his claim?

Essay Tips

There are two acronyms that are helpful with the three AP Lang writing branches.

Tip #1: SOAPS

Example text: Andrew Jackson’s speech to the Congress about sending Native Americans to the West.

Speaker: Identify the speaker of the piece, then analyze for bias and apply any prior knowledge that you have on the speaker.

Example: President Andrew Jackson had a bias against Native Americans. A piece written by Andrew Jackson about Native Americans will probably be written with a bias against him.

Occasion: Determine the time and the place of the written text, then identify the reason the text was written. Even if you aren’t sure of the reason, assume one and make your claim around it.

Example: Andrew Jackson was in office from 1829 to 1837. At this time, the Congress sent Native Americans to the West in order to clear the land for the colonists. Jackson was the one who made the proposal.

Audience: Who was the text directed to?

Example: Andrew Jackson’s speech was directed to a council.

Purpose: What is the text trying to say? Here, you analyze the tone of the text.

Example: Andrew Jackson appeals to pathos by calling Indians “savages”. His purpose is to portray Native Americans in a negative light, so the Congress passes the Indian Removal Act.

Subject: What is the main idea? What is the claim?

Example: Andrew Jackson wants the Congress to pass the Indian Removal Act because he believes Native Americans are uncultured and savage people.

Tip #2: Logos, Ethos, and Pathos

As you’ve probably learned before, Logos appeals to reason, Pathos appeals to emotion, and Ethos appeals to moral philosophy or credibility. However, for the AP Lang exam requires a wider understanding of the three.

If the text uses facts, statistics, quotations, and definitions, the speaker is appealing to Logos. Constituting various backup information is an extremely effective for people who want to persuade.

If the text uses vivid imagery and strong language it denotes Pathos, which is used to connect the audience to a piece emotionally; it is hardest to change the mind of a person who is linked to a subject via a strong emotion.

If the text attempts to demonstrate the speakers reliability or credibility, it is a direct appeal to Ethos. Using the example above, Andrew Jackson could have appealed to Ethos by stating the fact that he is the President of the United States, and thus, knows what is best for the union.

Often, Logos, Ethos, and Pathos lead to the use of logical fallacies.

Tip #3: DIDLS

This is a good shorthand for all textual analysis. While reading a text, try to pinpoint Diction, Imagery, Details, Language, and Sentence Structure in a piece. If anything stands out, add it to your analysis.


  • High range essay (8-9 points)
  • Effectively develops a position on the assigned topic.
  • Demonstrates full understanding of the sources or text.
  • Correctly synthesizes sources and develops a position. The writer drives the argument, not the sources.
  • The writer’s argument is convincing.
  • The writer makes no general assertions and cites specific evidence for each point. His/her evidence is developed and answers the “so what?” question.
  • The essay is clear, well-organized, and coherent. It is a stand alone piece rather than an exam response.
  • Contains very few grammatical and spelling errors or flaws, if any.

Note: 8-9 essays are an extreme rarity. A strong ‘7’ paper can jump to an 8-9 if the writing style is mature and perceptive.

Middle-Range Essay (57)

  • Adequately develops a position on the assigned topic.
  • Demonstrates sufficient understanding of the ideas developed in sources
  • Sufficiently summarizes the sources and assumes some control of the argument. ‘5’ essays are less focused than ‘6’ and ‘7’.
  • The writer's argument is sufficient but less developed.
  • Writer successfully synthesizes the sources and cites them.
  • Writer answers the “So what?” question but may use generalizations or assertions of universal truth. Writer cites own experience and specific evidence.
  • Essay is clear and well organized. ‘5’ essays less so.
  • Contains few minor errors of grammar or syntax.

Note: A ‘7’ is awarded to papers of college-level writing.
A ‘5’ on one of the AP English Language and Composition essays designates a 3 on the AP exam. It most likely relies on generalizations has limited control of the claim and argument. ‘5’ essays often lose focus and digress.

Low-Range Essays (1-4)

  • Inadequately develops a position on the assigned topic.
  • The author misunderstands and simplifies the ideas developed in the sources.
  • Over-summarizes the sources, lets the sources drive the argument.
  • Writer has weak control of organization and syntax. Essay contains numerous grammatical/spelling errors.
  • Writer does not cite the sources correctly, skips a citation, or cites fewer than the required minimum of the sources.
  • Notes: ‘4’ or ‘3’ essays do assert an argument but do not sufficiently develop it.
  • A ‘2’ essay does not develop an argument.
  • A 1-2 essay has severe writing errors and do not assert a claim.

Synthesis Essay Example

Essay Writing Advice From Our Professional Team

James Owen, online essay writer from EssayPro

The article reviews the basics of how to write a synthesis essay as well as how to dissect and analyze text when writing an AP English essay. One thing I would like to reemphasize is the importance of your thesis statement. When you write an essay for class or exam, make sure to state your argument clearly. If the reader of your essay doesn’t understand your point of view then what you’ve written is futile.

My advice is: when writing an essay in a short period (such as in an exam room) make sure to articulate your argument in every paragraph and connect every single one of your ideas to the thesis. My tip is to write your thesis down on a piece of paper and reread it at every point to ensure that the information applies and reinforces what you’ve stated in your thesis. This tip also goes for when you are writing a longer piece of writing, as it is very easy to lose focus and stray away from your main point.

Struggling With Writing an Essay?

Still having trouble crafting a synthesis essay? Need editing or writing help? You should seek advice from professional writers. Here at EssayPro, writers have written countless papers and are experts in their field. You can request to write your paper or editing or proofreading assistance. Rest assured that your paper is in good hands!


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