Cyclic stretch increases VEGF expression in pulmonary arterial smooth muscle cells via TGF-
1 and reactive oxygen species: a requirement for NAD(P)H oxidase
Eugenia Mata-Greenwood,1
Albert Grobe,2,3
Sanjiv Kumar,2,3
Yelina Noskina,1 and
Stephen M. Black2,3
1Department of Pediatrics, Northwestern University, Chicago, Illinois; and 2Department of Biomedical and Pharmaceutical Sciences and the 3International Heart Institute of Montana, Missoula, Montana
Submitted 4 November 2004
; accepted in final form 29 March 2005
 |
ABSTRACT
|
---|
Our previous studies have indicated that transforming growth factor (TGF)-
1 and VEGF expression are increased in the smooth muscle cell (SMC) layer of the pulmonary vessels of lambs with pulmonary hypertension secondary to increased pulmonary blood flow. Furthermore, we found that TGF-
1 expression increased before VEGF. Because of the increased blood flow in the shunt lambs, the SMC in the pulmonary vessels are exposed to increased levels of the mechanical force, cyclic stretch. Thus, in this study, using primary cultures of pulmonary arterial SMC isolated from pulmonary arteries of 4-wk-old lambs, we investigated the role of cyclic stretch in the apparent coordinated regulation of TGF-
1 and VEGF. Our results demonstrated that cyclic stretch induced a significant increase in VEGF expression both at the mRNA and protein levels (P < 0.05). The increased VEGF mRNA was preceded by both an increased expression and secretion of TGF-
1 and an increase in reactive oxygen species (ROS) generation. In addition, a neutralizing antibody against TGF-
1 abolished the cyclic stretch-dependent increases in both superoxide generation and VEGF expression. Our data also demonstrated that cyclic stretch activated an NAD(P)H oxidase that was TGF-
1 dependent and that NAD(P)H oxidase inhibitors abolished the cyclic stretch-dependent increase in VEGF expression. Therefore, our results indicate that cyclic stretch upregulates VEGF expression via the TGF-
1-dependent activation of NAD(P)H oxidase and increased generation of ROS.
vascular endothelial growth factor; transforming growth factor
BIOMECHANICAL FORCES ARE KNOWN to play an important role in maintaining the normal tissue architecture of the cardiopulmonary system (17). However, under certain pathological conditions, such as some types of congenital heart disease, this mechanical stimulation is increased significantly, and this may lead to various irreversible pathological conditions, such as pulmonary hypertension associated with vascular remodeling of the small pulmonary arteries (19, 20). We have previously shown that the development of pulmonary hypertension in lambs with increased pulmonary blood flow is associated with increased expression of the growth factors transforming growth factor (TGF)-
1 and VEGF (19, 20). VEGF is considered one of the most specific and potent angiogenic molecules. It has been shown to be essential for vascular development and normal processes such as wound healing and endometrium regeneration (6). However, VEGF expression can become dysregulated in certain pathological conditions such as tumorigenic growth and atherosclerosis (5, 6). Recent studies have also shown an increase in VEGF expression in various pulmonary hypertensive disorders, as in advanced pulmonary vascular disease secondary to congenital heart disease (5, 6). Various in vivo studies using models of increased blood flow have reported increases in VEGF expression (41). In vitro studies have confirmed that increased biomechanical forces can produce increased VEGF expression in various cell types (8, 22, 28, 30, 33). Indeed, the biomechanical force cyclic stretch has previously been shown to increase VEGF in pulmonary smooth muscle cells (26), whereas TGF-
1 has been reported to mediate this same effect in cardiomyocytes (41). In addition, VEGF expression has also been found to be sensitive to the cell redox status (15), and both superoxide and hydrogen peroxide can upregulate VEGF expression (13, 21, 29). Moreover, cyclic stretch has been shown to increase superoxide generation via an NAD(P)H oxidase (2). However, although cyclic stretch has been shown to increase the expression of the growth factors TGF-
1 and VEGF and activate NAD(P)H oxidase, little is known as to the connectivity between these events in the pulmonary vasculature.
In our animal model of increased pulmonary blood flow secondary to an aorta-pulmonary shunt, we observed an increase in VEGF expression in the media layer of remodeled vessels (19). This increase in VEGF expression occurred in the later phases of the disease, when vascular remodeling was evident (19, 20). In addition, we observed an earlier increase in TGF-
1 expression in the small pulmonary arteries of shunted lambs that preceded the VEGF upregulation (20). Thus we hypothesized that the increase in VEGF observed in our shunt model may be due, at least in part, to a previous increase in TGF-
1 expression and activation in the pulmonary vessels. To test this hypothesis, we utilized primary cultures of pulmonary artery smooth muscle cells (PASMC) isolated from 4-wk-old lambs exposed to cyclic stretch. We then analyzed the expression of VEGF and TGF-
1. VEGF expression was analyzed in the presence of a neutralizing antibody against TGF-
1. In addition, the involvement of reactive oxygen species (ROS) as an intermediary between TGF-
1 signaling and increased VEGF expression was investigated. The results obtained indicated that cyclic stretch-dependent increases in VEGF expression are dependent on TGF-
1-mediated activation of the NAD(P)H oxidase complex and increased generation of ROS. Thus therapies that modify ROS levels may be useful to prevent or limit the vascular remodeling associated with congenital heart disease and pulmonary hypertension secondary to increased pulmonary blood flow.
 |
MATERIALS AND METHODS
|
---|
Cell culture.
Primary cultures of PASMC from 4-wk-old sheep were isolated by the explant technique as we have described (39). Briefly, a segment of the main pulmonary artery from a 4-wk-old lamb was excised and placed in a sterile 10-cm dish containing DMEM supplemented with 1 g/l glucose. The segment was stripped of adventitia with a sterile forceps. The main pulmonary artery segment was then cut longitudinally to open the vessel, and the endothelial layer was removed by gentle rubbing with a cell scraper. The vessel was then cut into 2-mm segments, inverted, and placed on a collagen-coated 35-mm tissue culture dish. A drop of DMEM containing 10% FBS (Hyclone), antibiotics, and antimycotics (MediaTech) was then added, and the cells were grown overnight at 37°C in a humidified atmosphere with 5% CO2-95% air. The next day, an additional 2 ml of complete medium was added. The growth medium was subsequently changed every 2 days. When smooth muscle cell (SMC) islands were observed under the microscope, the tissue segment was removed, and the individual cell islands were subcloned. Identity was confirmed as PASMC by immunostaining (>99% positive) with SMC actin, caldesmon, and calponin. This was taken as evidence that cultures were not contaminated with fibroblasts or endothelial cells. All culture for subsequent experiments was maintained in DMEM supplemented with 10% FBS, antibiotics, and antimycotics at 37°C in a humidified atmosphere with 5% CO2-95% air. All experiments were conducted in cells between passages 5 and 15. The laboratory animal care committee at Northwestern University approved all protocols.
Cyclic stretch and cell treatments.
PASMC were exposed to cyclic stretch (of 1 Hz) using an FX-4000 Flexercell Tension Plus System. The FX-4000 Flexercell Tension Plus System consists of a four-place base plate equipped with 25-mm loading posts connected to the tension controller and vacuum pump with attached pressure/vacuum reservoir. The entire system is controlled by version 4.0 software allowing the various parameters to be adjusted, and frequency, % elongation, and duration of stretch can be programmed. Up to four plates can be stretched concurrently.
Preliminary studies indicated that a stretch of 20% of the total surface area increase was sufficient to induce VEGF, whereas 10% or lower failed to induce a significant upregulation of VEGF. Therefore, a cyclic stretch of 20% of total surface area was applied, using vacuum for various time periods (024). The frequency of stretching was 1 Hz (60 cycles/min), and we utilized a vacuum application to the flexible membrane resulting in a biaxial stretching over loading posts.
In the experimental protocols, PASMC were transferred to collagen-coated Bioflex plates and allowed to adhere for 18 h to produce confluency. Subsequently, the culture media containing 10% FBS were replaced with serum-free media, plus or minus NADPH oxidase inhibitors [1 µM diphenyleneiodonium (DPI), 10 µM 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF), or 10 µM apocynin], TGF-
1 neutralizing antibody (15 µg/ml, Calbiochem), or an anti-TGF-
2 receptor (10 µg/ml). All treatments were initially carried out using various concentrations of the inhibitors or neutralizing antibody, and the concentration where the maximum effect was observed without affecting control levels or inducing cytotoxicity was chosen. To confirm the lack of cytotoxicity, cells were stained for viability using the Live-Dead kit (Molecular Probes) according to the manufacturer's instructions. After being stained, the cells were rinsed with Dulbecco's PBS and visualized, and five random fields per sample were photographed for both the calcein (green, Live) and the ethidium (red, Dead) to determine viability.
In all our studies on VEGF expression, the cell media (conditioned media) were collected for ELISA analyses, whereas the cells were harvested for RNA analysis using semiquantitative RT-PCR. Thus results on protein and RNA expression are from matched samples.
Cloning, expression, and purification of ovine VEGF.
We cloned a sequence corresponding to +1 to +429 bp of the oVEGF120 (EMBL X89506
[GenBank]
). The following forward and reverse primers were used to create NdeI and HindIII restriction sites at the 5'- and 3'-ends, respectively: 5'-ACTAGTGGACATATGAACTTTCTGCTCTC-3' and 5'-ACCGCCTAAGCTTGTCACATTTTTCTTG-3'. The 429-bp product was cloned into the pET23b bacterial expressing vector (Novagen) to include the His-tag sequence at the COOH terminus. The sequence was verified by Northwestern Biotechnology Laboratories. Rosetta (DE3) cells (Novagen) were transformed with this construct and then grown in Circle Growth Media at 37°C for 3 h and then at 21°C for 8 h in the presence of 100 µM isopropylthiogalactoside (Novagen). His-tagged ovine VEGF was then purified using nickel-charged columns (Pierce) and biotinylated using Sulfo-NHS-LC-Biotin (Pierce) and then repurified using a Dextran desalting column using the manufacturer's suggested protocols.
TGF-
1 and VEGF ELISAs.
Active and latent TGF-
1 concentrations in cell culture supernatants were determined using commercially available ELISA from R&D Systems. Sensitivity with this kit is 31.5 ng/ml. Latent TGF-
1 was activated by treatment with 0.2 volumes of 1 N HCl for 10 min followed by neutralization with 2 N NaOH in HEPES buffer. All procedures followed the manufacturer's protocol. Analyses were performed in duplicate from at least three independent experiments, and a standard TGF-
1 curve was used in the all cases. The color of the chromogenic reaction was evaluated spectrophotometrically at 450 nm using a plate reader (Labsystems).
To determine the concentration of VEGF secreted in cell culture supernatants, we developed a competitive ELISA protocol, since the commercially available kits did not adequately recognize ovine VEGF. A 96-well RIA plate was coated at room temperature overnight with polyclonal rabbit anti-ovine VEGF (19). After three washes with 400 µl of 0.5% Tween in PBS (wash buffer), 300 µl of blocking solution (1% BSA, 5% sucrose in wash buffer) were added to each well and were allowed to incubate for at least 1 h at room temperature. Each well was aspirated and washed three times with 400 µl of wash buffer as described previously (19). Then, 100 µl of cell supernatant and 50 µl of biotinylated ovine VEGF (0.5 ng/µl) were added, in duplicate fashion. After a 2-h incubation period, each well was carefully aspirated and again washed three times with wash buffer. Biotinylated ovine VEGF was visualized as described for the R&D Systems ELISA kit protocol. A positive standard curve was obtained by using nonbiotinylated ovine VEGF in various concentrations (0.525 ng/well). Cross-reactivity with basic fibroblast growth factor, TGF-
1, and PDGF was determined to be <5% (data not shown).
Semiquantitative RT-PCR.
Total RNA was collected using the TRIzol reagent. After extraction with chloroform and purification with isopropanol and ethanol, total RNA was quantified spectrophotometrically. Total RNA (0.1 µg) was reverse transcribed and amplified using a single-step RT-PCR kit (Invitrogen). The final reaction mixture contained 1.2 mM MgSO4, 0.5 mM DTT, 0.2 µM dNTP, 1 unit of RT/Platinum Taq mixture and 0.2 µM of each specific primer. The following cycling conditions were established using a DNA Thermal Cycler: 1 cycle at 50°C for 45 min (RT step) followed by a denaturation step at 94°C for 2 min, and a set of 1525 cycles of: 94°C for 30 s, 55°C for 60 s, and 68°C for 90 s. In all cases, cycle curves were prepared to determine the number of cycles that produced linear amplification of the gene of interest. These were determined to be: 15 cycles for 18S (internal control) and 25 cycles for VEGF and TGF-
1. In all cases, the 18S amplification occurred in the same reaction tube as either VEGF or TGF-
1. The primer sets used in The RT-PCR analyses were as follows: ovine VEGF120 sense primer: 5'-ATTGGAGCCTTGCCTTGCTG-3' and antisense primer: 5'-ACTCATCTCTCCTATGTGCTGG-3' yielding a product of 335 bp (11); TGF-
1 sense: 5'-CTTCCTGCTCCTCATGGCCACC-3' and antisense primer: 5'-CAGGAGCGCACGATCATGTTGGACA-3' yielding a product of 393 bp (corresponding to bp 7681160 from X 76916 Ovis aries TGF-
1 mRNA sequence); and 18S sense primer: 5'-AGGGTTCGATTCCGGAGAGGG-3' and antisense primer: 5'-CATTCCAATTACAGGGCCTCG-3' yielding a product of 146 bp.
Estimation of cellular ROS levels.
Changes in cellular ROS levels were estimated using the oxidation of dihydroethidium (DHE) to ethidium as we have described previously (39). Briefly, cells were seeded and allowed to adhere for at least 18 h. Cells were then washed in PBS and incubated in serum-free DMEM. Cells were then exposed to cyclic stretch or TGF-
1 was added as required and in the presence and absence of various NADPH oxidase inhibitors. DHE (10 µM, Molecular Probes) was added to the medium 15 min before the end of the experiment. Cells were washed with PBS and imaged using a Nikon Eclipse TE-300 fluorescence microscope. DHE-stained cells were observed after excitation at 518 nm and emission at 605 nm. Fluorescence images were captured with a Photometrix digital camera, and the average fluorescence intensities (to correct for differences in cell number) were quantified using Metamorph imaging software (Fryer). Statistical analyses between treatments were carried out as detailed (see Statistical analysis).
Analysis of p47phox translocation.
PASMC exposed to TGF-
1 or exposed to cyclic stretch were lysed using a commercial cold lysis buffer (Upstate Biotechnology) containing a protease inhibitor cocktail (Sigma). After sonication, cell extracts were centrifuged at 100,000 g for 45 min at 4°C. The membrane and soluble fractions were then collected and the membrane fraction was resuspended in cold lysis buffer and sonicated again to solubilize the membrane proteins. Protein extracts (20 µg) were then separated on 12% SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T). After a 1-h blocking, the membranes were incubated at 4°C overnight with 1:200 dilution of rabbit-polyclonal anti-p47phox antibody (Santa Cruz). Membranes were washed 3x 15 min with TBS-T and then incubated for 45 min with an anti-rabbit IgG conjugated to horseradish peroxidase. After a further 3x 15-min wash in TBS-T, bands were visualized with chemiluminescence using a Kodak Digital Science Image Station (NEN) as described previously (1).
Determination of cellular NADH and NADPH oxidation.
NADPH and NADH oxidation were followed spectrophotometrically at 340 nm in a Biospec 1601 (Shimadzu) as previously described (31). The reactions were carried using the particulate fraction of the PASMC (0.02 mg/ml) diluted to a final volume of 1 ml in phosphate buffer (pH 7.4). NADPH or NADH (0.1 mM) were added just before the measurements were started and followed for 5 min. The experiments were carried out at room temperature. The rate of NADPH and NADH oxidation was calculated using a molar extinction coefficient of 6.22 mM1cm1 (31).
Statistical analysis.
In all experiments, the means ± SD were calculated, and comparisons were made by ANOVA. When differences were present between study groups, Student-Newman-Keuls post hoc testing was performed. P < 0.05 was considered statistically significant.
 |
RESULTS
|
---|
Cyclic stretch upregulates VEGF expression in 4-wk-old PASMC.
We initially examined the effect of cyclic stretch on PASMC VEGF mRNA expression using semiquantitative RT-PCR and protein secretion using an ELISA assay we developed. Our results indicated that cyclic stretch, at 20% of surface area, significantly increased VEGF mRNA levels as early as 4 h (Fig. 1, A and B, P < 0.05 vs. static control). VEGF mRNA levels were also elevated relative to static controls at 8 h, although the relative increase in expression was reduced compared with that seen at 4 h (Fig. 1, A and B, P < 0.05 vs. static control). In accordance with changes in the transcript levels, VEGF protein secretion was increased by 4 and 8 h of cyclic stretch (20% of surface area) compared with static controls (Fig. 1C, P < 0.05 vs. static control). The increase in VEGF was found to be dependent on the level of cyclic stretch applied, as 4 and 10% stretch of surface area did not increase VEGF expression, whereas 30% increased VEGF expression to a similar extent as 20% but was also associated with a decrease in cell number (data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1. Cyclic stretch upregulates VEGF expression in 4-wk-old pulmonary arterial smooth muscle cells (PASMC). Time course studies were performed using confluent 4-wk-old PASMC grown on collagen-coated Bioflex plates. Cells were exposed to cyclic stretch (08 h) representing 20% of surface area as described in MATERIALS AND METHODS, and then total RNA and media were isolated for mRNA and protein determinations, respectively. A: total RNA was isolated from PASMC and subjected to RT-PCR analysis using primers to specifically amplify both ovine VEGF and ovine 18S (as an internal control), and the products were separated by agarose gel electrophoresis. A representative RT-PCR for VEGF and 18S is shown. B: densitometric values for relative VEGF mRNA (normalized to 18S mRNA) from 6 independent experiments. Cyclic stretch significantly increases VEGF mRNA levels after 4 and 8 h. Values are means ± SD. *P < 0.05 vs. static cells. C: effect of stretch on VEGF protein secretion. The collected media were analyzed by ELISA to determine the levels of secreted VEGF. Results are expressed as the percentage of VEGF secreted relative to the static control at that time point. Data are means ± SD from 6 independent experiments. *P < 0.05 vs. static cells.
|
|
Cyclic stretch upregulates expression of TGF-
1 but not its activation.
Cyclic stretch has been reported to induce the expression of TGF-
1 in other cell culture systems (18, 24, 41). Thus, we examined the effect of cyclic stretch, at 20% of surface area, on TGF-
1 expression and activation. ELISA analysis indicated that cyclic stretch produced a significant increase in total TGF-
1 secretion (Fig. 2A, P < 0.05 vs. static control). However, this increase was limited to the latent form of TGF-
1, and no increase in the percentage of TGF-
1 activation was detected (Fig. 2B). Semiquantitative RT-PCR analysis demonstrated that 2 h of cyclic stretch, at 20% of total surface area, increased TGF-
1 mRNA compared with static controls (Fig. 2C, P < 0.05 vs. static control). However, this increase was transient, and after 4 and 8 h of cyclic stretch, TGF-
1 mRNA levels were similar to the static controls (Fig. 2C).
Cyclic stretch-induced increase in VEGF expression is TGF-
1 dependent.
A previous study by Zheng et al. (41) indicated that TGF-
1 upregulation was essential for subsequent stretch-induced VEGF expression in cardiomyocytes. Therefore, we determined whether the cyclic stretch-dependent increases in VEGF expression in PASMC had the same requirement for TGF-
1. To accomplish this, we examined the effect of a neutralizing TGF-
1 antibody on the cyclic stretch-induced increase in VEGF expression. The results obtained indicated that the upregulation of VEGF induced by cyclic stretch was abolished by the neutralizing antibody to TGF-
1 (Fig. 3, AC, P < 0.05 vs. cyclic stretch alone). This effect was only seen at concentrations of
4 µg/ml, and concentrations of
4 µg/ml failed to abolish the cyclic stretch-dependent increase in VEGF expression (data not shown). This antibody also decreased basal (static control) expression of VEGF mRNA (P < 0.05 vs. untreated static control), suggesting that TGF-
1 may also have an important role in regulating basal VEGF expression in PASMC. Similarly, a polyclonal antibody raised against the TGF-
2 receptor prevented the cyclic stretch-dependent increase in VEGF protein in PASMC (Fig. 3D).
Cyclic stretch increases superoxide generation via TGF-
1.
Cyclic stretch (9, 10, 16) and TGF-
1 (3, 16, 3437) have previously been shown to induce the generation of ROS in various cellular phenotypes. Therefore, we next analyzed the effect of cyclic stretch on ROS generation in PASMC. We used the oxidation of DHE to ethidium to estimate cellular ROS levels. Our results demonstrated that cyclic stretch increased ethidium fluorescence in PASMC 2.4-fold (Fig. 4, A and B, P < 0.05 vs. static control). This increase was time dependent with no increase in ROS levels detected when cells were exposed to <2 h of exposure to cyclic stretch (data not shown). It should be noted that these experiments were carried out in media containing 10% FBS, as ethidium fluorescence was below the level of detection in serum-free medium. We also found that the increase in ethidium fluorescence was TGF-
1 dependent, as the addition of a TGF-
1 neutralizing antibody (4 µg/ml) decreased the levels of ethidium fluorescence in both static and stretched PASMC (Fig. 4, A and B, P < 0.05 vs. untreated). To confirm the importance of TGF-
1, PASMC treated with TGF-
1 (5 ng/ml, 4 h) exhibited a 1.9-fold increase in ethidium fluorescence, indicating increased ROS levels (Fig. 4C, P < 0.05 vs. untreated PASMC).
Cyclic stretch activates NAD(P)H oxidase.
Various reports have indicated the presence of NAD(P)H oxidase subunits in vascular smooth muscle cells, including p22phox, p47phox, p67phox, and functionally active gp91phox (12, 38). Moreover, it has been suggested that TGF-
1-induced generation of ROS may involve the NAD(P)H oxidase complex (3437). Thus we next investigated whether TGF-
1- or cyclic stretch-mediated increases in ROS levels in PASMC occurred through NAD(P)H oxidase activation. To begin to examine this possibility, we initially determined whether cyclic stretch and/or TGF-
1 induced the translocation of p47phox to the plasma membrane. Utilizing Western blot analysis of cytosolic and plasma membrane fractions, we found that <10% of the total p47phox was present in control (static) plasma membranes (Fig. 5, A and B). However, after exposure to cyclic stretch (20% of surface area, 2 h), 55% of the p47phox protein was found to translocate to the membrane (Fig. 5, A and B, P < 0.05 vs. static control). Again, the addition of a neutralizing antibody against TGF-
1 (4 µg/ml) completely abolished the cyclic stretch-induced translocation of p47phox to the membrane (Fig. 5, A and B; P < 0.05 vs. cyclic stretch). Moreover, treatment of PASMC with TGF-
1 also significantly increased the translocation of p47phox to the membrane of PASMC (Fig. 5, A and B, P < 0.05 vs. static control). Further evidence that TGF-
1 increases PASMC ROS levels via NADPH oxidase activation was obtained by examining ethidium fluorescence in the presence of NADPH oxidase inhibitors with different mechanisms of action. Apocynin, a reversible and specific inhibitor of NADPH oxidase (32), DPI, an irreversible inhibitor of NADPH oxidase (7), and AEBSF, a serine protease inhibitor that can inhibit NADPH oxidase (4), were used. All three inhibitors blocked the increase in ROS levels induced by TGF-
1 (Fig. 6, P < 0.05 vs. TGF-
1 alone). We found that DPI significantly increased basal ROS levels (Fig. 6, P < 0.05 vs. untreated) but prevented the increase in ROS associated with the addition of TGF-
1 (Fig. 6). We have previously shown that DPI can increase ROS levels in SMC by producing a loss of mitochondrial membrane potential, but it prevented endothelin-1-dependent activation of NADPH oxidase (39). Because this could also be associated with a loss of cell viability, we also carried out a live-dead cell analysis to determine if either cyclic stretch itself or any of the inhibitors used was altering PASMC viability. However, the data obtained indicated that there was no increase in cell death and no reduction in cell viability by any of the treatments used (Fig. 7).

View larger version (72K):
[in this window]
[in a new window]
|
Fig. 7. Cyclic stretch or NADPH oxidase antagonists do not alter the viability of 4-wk-old PASMC. Studies were performed using 4-wk-old PASMC grown on collagen-coated Bioflex plates. Cells were treated with AEBSF (10 µM), allopurinol (100 µM), apocynin (1 mM), DPI (1 µM), or anti-TGF- 1 (4 µg/ml) in serum-free DMEM. The cells were subjected to cyclic stretch at 1 Hz with 20% elongation for 4 h. Parallel samples were incubated for 4 h without stretch. At the end of the 4 h, the cells were stained for viability using the Live-Dead kit. After being stained, the cells were rinsed with Dulbeccos PBS and visualized, and 5 random fields/sample were photographed for both the calcein (green, Live) and the ethidium (red, Dead) to determine viability. No significant loss of viability was seen over the period of 4 h with or without stretch and inhibitors. A: calcein (Live) and B: ethidium (Dead), no stretch; C: calcein (Live) and D: ethidium (Dead), stretch. Image is representative of 3 independent experiments.
|
|
Next we determined whether the translocation of the p47phox subunit to the plasma membrane correlated with the activation of the NAD(P)H oxidase complex. To accomplish this, we measured the cellular consumption of NADH and NADPH. Our results indicated that cyclic stretch significantly increased the consumption of NADPH (P < 0.05 vs. static control) with no alterations in NADH consumption (Table 1). Again, the addition of a neutralizing antibody to TGF-
1 significantly reduced the cyclic stretch-dependent increase in NADPH consumption (P < 0.05 vs. cyclic stretch, Table 1). Interestingly, the addition of TGF-
1 increased the consumption of both NADPH and NADH (P < 0.05 vs. static control, Table 1). Pretreatment with DPI before cell harvesting inhibited NADPH consumption under all conditions, again suggesting that the most significant consumer of NADPH in PASMC is the NADPH oxidase complex (Table 1).
Cyclic stretch-induced superoxide generation via activation of a NADPH oxidase complex is required for VEGF upregulation.
We next analyzed the effect of the NADPH oxidase inhibitors apocynin, DPI, and AEBSF on the upregulation of VEGF by cyclic stretch. Cyclic stretch-induced VEGF expression was blocked by all three inhibitors (Fig. 8). In addition, we found that apocynin and DPI did not alter basal levels of VEGF but prevented the cyclic stretch-mediated increase in VEGF expression (Fig. 8, P < 0.05 vs. stretch alone), whereas AEBSF decreased both basal and cyclic stretch-induced VEGF expression (Fig. 8, P < 0.05 vs. stretch alone). Allopurinol (100 µM), a xanthine oxidase inhibitor, did not alter the cyclic stretch-induced VEGF expression in PASMC (Fig. 8).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 8. Effect of NAD(P)H oxidase inhibitors on the cyclic stretch-induced increase in VEGF expression in 4-wk-old PASMC. PASMC were exposed to cyclic stretch (20% of surface area, 4 h) in the presence or absence of the NAD(P)H oxidase inhibitors DPI (1 µM), AEBSF (10 µM), or apocynin (1 mM), or the xanthine oxidase inhibitor allopurinol (100 µM). Total RNA and media were then isolated for mRNA and protein determinations, respectively. A: total RNA isolated from PASMC was subjected to RT-PCR analysis using primers to specifically amplify both ovine VEGF and ovine 18S (as an internal control), and the products were separated by agarose gel electrophoresis. A representative RT-PCR for VEGF and 18S is shown. B: densitometric values for relative VEGF mRNA (normalized to 18S mRNA). The cyclic stretch-dependent increase in VEGF mRNA levels was inhibited by NADPH oxidase inhibition but not by xanthine oxidase inhibition. Values are means ± SD from 4 independent experiments. *P < 0.05 vs. control (untreated static cells), P < 0.05 vs. cyclic stretch alone. C: effect of stretch on VEGF protein secretion. The collected media were analyzed by ELISA to determine the levels of secreted VEGF protein. Results are expressed as relative VEGF-secreted protein ± SD compared with control (untreated static cells) levels. NADPH oxidase inhibition, but not xanthine oxidase inhibition, prevents the cyclic stretch-dependent increase in VEGF secretion. Data are from 4 independent experiments. *P < 0.05 vs. control (untreated static cells), P < 0.05 vs. stretch alone. P < 0.05 vs. control static.
|
|
Finally, we analyzed the effect of the NAD(P)H oxidase inhibitors on TGF-
1-mediated increase of VEGF protein levels in PASMC. The results obtained indicated that DPI and apocynin did not affect basal levels of VEGF protein but again blocked the TGF-
1-mediated increase in VEGF expression (Fig. 9, P < 0.05 vs. TGF-
1 alone). In contrast with the effect on cyclic stretch-induced VEGF expression, AEBSF had little blocking effect on TGF-
1-mediated increase (Fig. 9). To confirm that the effects observed were specific to NAD(P)H oxidase activity and not to other superoxide-generating enzymes, we again utilized the xanthine oxidase inhibitor allopurinol. Allopurinol (100 µM) did not block the upregulation of VEGF induced by TGF-
1 treatment (Fig. 9).
 |
DISCUSSION
|
---|
Our previous studies using an in vivo model of pulmonary hypertension secondary to increased pulmonary blood flow have shown that TGF-
1 and VEGF expression become dysregulated in a sequential fashion (19, 20). Thus the purpose of this study was to further investigate potential causal links between the two growth factors and to examine the role of the biomechanical force, cyclic stretch, in this process. For this purpose, we utilized an in vitro cell culture model using primary cultures of PASMC isolated from 4-wk-old lambs exposed to various degrees of cyclic stretch. The results obtained from these studies indicated that cyclic stretch produced an increase in VEGF expression within 4 h, and this effect was maintained up to 8 h, although the magnitude was decreased. This transient effect is in agreement with our in vivo data where VEGF expression was found to peak at 4 wk and was decreased at 8 wk of age (19). Interestingly, the time course of VEGF expression we determined was different with that observed in the recent study by Quinn et al. (26). In this study, the increased expression of VEGF was not detected until 12 h (26). The increase in VEGF was maintained at 24 h but was back at baseline by 48 h (26). The differences in temporal response is difficult to reconcile, although the study by Quinn et al. (26) utilized PASMC isolated from 6-wk-old lambs, and thus it is possible that there may be developmental differences in the response of PASMC to cyclic stretch producing a delay in the increase in VEGF expression. In addition, two other points are worth making here. The data presented by Quinn et al. utilized biaxial cyclical stretch (1 Hz) at an amplitude of 15% (26), whereas our studies utilized similar biaxial cyclical stretch at 1 Hz but with an amplitude of 20%. Thus it is possible that the decreased amplitude used in the Quinn et al. studies increases the time required to see significant increases in VEGF expression. Second, Quinn et al. utilized an older model of mechanical strain device, the Flexcell 3000 Strain Unit (Flexcell). This again could account for the temporal differences in increase in VEGF expression.
Our data also demonstrated that the increase in VEGF expression was preceded by an increase in TGF-
1 expression and secretion. Again, this is consistent with the sequential upregulation we have previously observed in our lamb model of increased pulmonary blood flow (19, 20). Considering that TGF-
1 has been reported in separate studies to increase VEGF expression and activate NAD(P)H oxidases in various cell systems (3, 35, 36), we investigated the contribution of increased TGF-
1 production in the increased VEGF expression in PASMC using a neutralizing antibody against TGF-
1. This antibody was found to abolish the cyclic stretch-induced increase in VEGF expression. The neutralizing antibody also decreased both the cyclic stretch- and TGF-
1-dependent increases in ROS generation and NAD(P)H oxidase activation in PASMC. In addition, the neutralizing antibody inhibited both basal levels of ROS and VEGF mRNA and secreted protein, suggesting that TGF-
1 may have an important homeostatic role in PASMC. Thus our observations on the effects of the neutralizing TGF-
1 antibody indicate that the increased TGF-
1 expression induced by cyclic stretch is an important upstream event leading to increased ROS and VEGF transcription. Furthermore, this suggests that an imbalance in the TGF-
1 system may contribute to the development of pulmonary hypertension.
A role for ROS in both the myogenic tone and in the mediation of mechanotransduction pathways in the vessel wall has been suggested by previous studies. Recent reports have shown that the myogenic response of arteries and arterioles can be inhibited by scavenging of ROS (14, 23). In addition, NAD(P)H oxidase activation and subsequent generation of ROS can lead to increased vascular smooth muscle contractility and proliferation (25, 39, 40). Our study demonstrates that cyclic stretch increases the levels of ROS by the activation of a NAD(P)H oxidase in PASMC. These data suggest that cell signaling induced by cyclic stretch, a physiologically relevant force in vivo, has a ROS component in pulmonary arteries. Moreover, we found that the activation of NAD(P)H oxidase and the subsequent increase in ROS generation were two events necessary for the subsequent upregulation of VEGF in PASMC.
Our data may have important implications regarding molecular targets in pulmonary hypertension arising from conditions other than increased pulmonary blood flow. For example, it has been shown that NAD(P)H oxidase inhibitors can decrease pulmonary vascular resistance due to hypoxia (7). Similarly, we have observed activation of NAD(P)H oxidases in an ovine model of persistent pulmonary hypertension of the newborn produced by ductal ligation, whereas a recombinant superoxide dismutase was capable of reversing many of the manifestations of this disease (1). Therefore, NAD(P)H oxidase might become activated under various conditions and participate in the pathogenesis of various pulmonary hypertensive disorders. Importantly, we observed that NAD(P)H oxidase inhibitors could block the increase in VEGF expression produced by cyclic stretch without affecting basal homeostasis, in contrast with our observations with the TGF-
1 blocking antibody. This suggests that specifically targeting the NADPH oxidase complex may have therapeutic potential for the treatment of pulmonary hypertensive disorders. However, more studies will be required to determine how TGF-
1 actually activates the NADPH oxidase complex.
It should be noted that our data suggest that there may differences between TGF-
1-treated and cyclic stretch-treated PASMC. Although both systems caused an increase in superoxide levels and VEGF expression and secretion, they responded differently to NAD(P)H oxidase inhibitors. For instance, AEBSF, a serine protease inhibitor that also inhibits NADPH oxidase activity (4), produced complete inhibition of cyclic stretch-induced VEGF expression, whereas it had only a weak effect on increases in VEGF expression produced by the addition of exogenous TGF-
1. This effect may be explained by the ability of AEBSF to inhibit plasmin, a serine protease inhibitor known to activate latent TGF-
1 (27). Because cyclic stretch induces the release of latent TGF-
1 (as shown by ELISAs) and subsequent progressive activation of TGF-
1 is needed for biological function, the inhibition of plasmin by AEBSF might contribute to the potent inhibition of cyclic stretch-induced VEGF expression. This could also explain why AEBSF did not completely abolish the increase in VEGF expression in PASMC induced by the addition of recombinant TGF-
1, although it completely blocked the production of ROS by this growth factor (as estimated by alterations in ethidium fluorescence). In addition, although both cyclic stretch and TGF-
1 induced p47phox translocation and ROS generation in PASMC, NADH/NADPH consumption rates were different. TGF-
1 increased the consumption of NADH to a higher degree and NADPH to a lesser degree, and cyclic stretch had the opposite effect. TGF-
1 has been shown to activate a NADH-dependent oxidase that generates H2O2 in fibroblasts, keratinocytes, and endothelial cells (36, 37). This activation is a delayed and prolonged event (424 h posttreatment) and has been shown to mediate the activation of MAP kinases and p38. This effect has been proposed as a mechanism by which TGF-
1 induces cellular proliferation (3, 3437), although the precise NAD(P)H oxidase system that is activated by cyclic stretch has not been well characterized. Our data are in agreement with these earlier studies as we observed a cyclic stretch-mediated translocation of p47phox and increased ROS levels that were inhibited by DPI. Interestingly, we did not observe an increase in NADH consumption in our stretch-stimulated PASMC in spite of the fact that this event is dependent on TGF-
1. This suggests that cyclic stretch may stimulate novel molecular pathways that lead to activation of NADPH and of the TGF-
1 and VEGF systems in a concerted manner. In our PASMC, the signaling events downstream of increased ROS that lead to increased VEGF expression remain to be elucidated, and further studies will be required to determine whether this increase is mediated by an increase in the transcription of the VEGF gene, and, if so, more studies will be needed to identify the oxidant-sensitive transcription factor involved in the transcriptional activation of VEGF. Because activator protein-1, hypoxia-inducible factor-1
, and NF-
B have all been shown to play important roles in VEGF transcriptional regulation, and all three have been shown to be activated by ROS, these will be obvious first targets (9, 10).
In conclusion, we have shown that cyclic stretch induces the expression of TGF-
1 that in turn activates an NADPH oxidase complex, thereby increasing ROS levels, and that these events are necessary for the subsequent increase in VEGF transcription in PASMC. Inhibition of NADPH oxidase inhibited cyclic stretch-induced, but not basal expression, of VEGF, whereas inhibition of TGF-
1 signaling (using a neutralizing antibody) inhibited both basal and stimulated VEGF expression. Our data suggest that basal expression of VEGF might be regulated by non-NADPH oxidase-dependent mechanisms, whereas stimulation by cyclic stretch leads to the activation of a TGF-
1-dependent signaling pathway via NADPH oxidase with increased ROS generation as a signaling intermediary. Furthermore, our data indicate that TGF-
1 has an important role in cyclic stretch activation of signaling pathways in PASMC and that dysregulation of this signaling pathway might contribute to the vascular remodeling associated with pulmonary hypertension secondary to increased pulmonary blood flow.
 |
GRANTS
|
---|
This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-60190, HL-67841, HL-072123, and HL-070061 (all to S. M. Black), March of Dimes 6-FY00-98, and American Heart Association Pacific Mountain Affiliates 0550133Z (to S. M. Black).
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. M. Black, International Heart Institute of Montana, 3rd Floor, St. Patrick Hospital, 554 West Broadway, Missoula, MT 59802 (e-mail: stephen.black{at}umontana.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
 |
REFERENCES
|
---|
- Brennan L, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, and Black SM. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. Circ Res 92: 683691, 2003.[Abstract/Free Full Text]
- Chen Q, Li W, Quan Z, and Sumpio BE. Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and p38 mitogen-activated protein kinase. J Vasc Surg 37: 660668, 2003.[CrossRef][ISI][Medline]
- Chiu C, Maddock DA, Zhang Q, Souza KP, Townsend AR, and Wan Y. TGF-
-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes. Int J Mol Med 8: 251255, 2001.[ISI][Medline]
- Diatchuk V, Lotan O, Koshkin V, Wikstroem P, and Pick E. Inhibition of NADPH oxidase activation by 4-(2-aminoethyl)-benzenesulfonyl fluoride and related compounds. J Biol Chem 272: 1329213301, 1997.[Abstract/Free Full Text]
- Ferrara N, Houck K, Jakeman L, and Leung DW. Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 13: 1832, 1992.[CrossRef][ISI][Medline]
- Frelin C, Ladoux A, and D'Angelo G. Vascular endothelial growth factors and angiogenesis. Ann Endocrinol 61: 7074, 2000.[ISI][Medline]
- Grimminger F, Weissmann N, Spriestersbach R, Becker E, Rosseau S, and Seeger W. Effects of NADPH oxidase inhibitors on hypoxic vasoconstriction in buffer-perfused rabbit lungs. Am J Physiol Lung Cell Mol Physiol 268: L747L752, 1995.[Abstract/Free Full Text]
- Gruden G, Thomas S, Burt D, Lane S, Chusney G, Sacks S, and Viberti G. Mechanical stretch induces vascular permeability factor in human mesangial cells: mechanisms of signal transduction. Proc Natl Acad Sci USA 94: 1211212116, 1997.[Abstract/Free Full Text]
- Hishikawa K and Luscher T. Pulsatile stretch stimulates superoxide production in human aortic endothelial cells. Circulation 96: 36103616, 1997.[Abstract/Free Full Text]
- Hishikawa K, Oemar BS, Yang Z, and Luscher T. Pulsatile stretch stimulates superoxide production and activates nuclear factor-
B in human coronary smooth muscle. Circ Res 81: 797803, 1997.[Abstract/Free Full Text]
- Jabbour H, Boddy SC, and Lincoln GA. Pattern and localization of expression of vascular endothelial growth factor and its receptor flt-1 in the ovine pituitary gland: expression is independent of hypothalamic control. Mol Cell Endocrinol 134: 91100, 1997.[CrossRef][ISI][Medline]
- Javaesghani D, Magder SA, Barreiro E, Quinn MT, and Hussain SNA. Molecular characterization of a superoxide-generating NAD(P)H oxidase in the ventilatory muscles. Am J Respir Crit Care Med 165: 412418, 2002.[Abstract/Free Full Text]
- Khanna S, Roy S, Bagchi D, Bagchi M, and Sen CK. Upregulation of oxidant-induced VEGF expression in cultured keratinocytes by a grape seed proanthocyanidin extract. Free Radic Biol Med 31: 3842, 2001.[CrossRef][ISI][Medline]
- Koller A. Signaling pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension. Microcirculation 9: 277294, 2002.[CrossRef][ISI][Medline]
- Kuin A, Kruse JJ, and Stewart FA. Proteinuria and vascular changes after renal irradiation: the role of reactive oxygen species (ROS) and vascular endothelial growth factor (Vegf). Radiat Res 159: 174181, 2003.[ISI][Medline]
- Lehoux S, Esposito B, Merval R, Loufrani L, and Tedgui A. Pulsatile stretch-induced extracellular signal-regulated kinase 1 activation in organ culture of rabbit aorta involves reactive oxygen species. Arterioscler Thromb Vasc Biol 20: 23662372, 2000.[Abstract/Free Full Text]
- Lehoux S and Tedgui A. Cellular mechanics and gene expression in blood vessels. J Biomech 36: 631643, 2003.[CrossRef][ISI][Medline]
- Lindahl G, Chambers RC, Papakrivopoulou J, Dawson SJ, Jacobsen MC, Bishop JE, and Laurent GF. Activation of fibroblast procollagen
1(I) transcription by mechanical strain is transforming growth factor-
-dependent and involves increased binding of CCAAT-binding factor (CBF/NF-Y) at the proximal promoter. J Biol Chem 277: 61536161, 2002.[Abstract/Free Full Text]
- Mata-Greenwood E, Meyrick B, Soifer SJ, Fineman JR, and Black SM. Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 285: L222L231, 2003.[Abstract/Free Full Text]
- Mata-Greenwood E, Meyrick B, Steinhorn RH, Fineman JR, and Black SM. Alterations in TGF-
1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 285: L209L221, 2003.[Abstract/Free Full Text]
- Maulik N and Das DK. Redox signaling in vascular angiogenesis. Free Radic Biol Med 33: 10471060, 2002.[CrossRef][ISI][Medline]
- Muratore C, Nguyen HT, Ziegler MM, and Wilson JM. Stretch-induced upregulation of VEGF gene expression in murine pulmonary culture: a role for angiogenesis in lung development. J Pediatr Surg 35: 906912, 2000.[CrossRef][ISI][Medline]
- Nowicki PT, Flavahan S, Hassanain H, Mitra S, Holland S, Goldschmidt-Clermont PJ, and Flavahan NA. Redox signaling of the arteriolar myogenic response. Circ Res 89: 114116, 2001.[Abstract/Free Full Text]
- O'Callaghan C and Williams B. Mechanical strain-induced extracellular matrix production by human vascular smooth muscle cells: role of TGF-
1. Hypertension 36: 319324, 2000.[Abstract/Free Full Text]
- Oeckler R, Kaminski PM, and Wolin MS. Stretch enhances contraction of bovine coronary arteries via a NAD(P)H oxidase-mediated activation of the extracellular signal-regulated kinase mitogen-activated protein kinase cascade. Circ Res 92: 2331, 2003.[Abstract/Free Full Text]
- Quinn TP, Schlueter M, Soifer SJ, and Gutierrez JA. Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 282: L897L903, 2002.[Abstract/Free Full Text]
- Sato Y, Tsuboi R, Lyons H, Moses H, and Rifkin DB. Characterization of the activation of latent TGF-
by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system. J Cell Biol 111: 757763, 1990.[Abstract]
- Seko Y, Takahashi N, Shibuya M, and Yazaki Y. Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes. Biochem Biophys Res Commun 254: 462465, 1999.[CrossRef][ISI][Medline]
- Sen CK, Khanna S, Babior BM, Hunt TK, Ellison EC, and Roy S. Oxidant-induced vascular endothelial growth factor expression in human keratinocytes and cutaneous wound healing. J Biol Chem 277: 3328433290, 2002.[Abstract/Free Full Text]
- Smith J, Davies N, Willis AL, Sumpio BE, and Zilla P. Cyclic stretch induces the expression of vascular endothelial factor in vascular smooth muscle cells. Endothelium 8: 4148, 2001.[Medline]
- Souza H, Liu X, Amouilov A, Kuppusamy P, Laurindo FR, and Zweier JL. Quantitation of superoxide generation and substrate utilization by vascular NAD(P)H oxidase. Am J Physiol Heart Circ Physiol 282: H466H474, 2002.[Abstract/Free Full Text]
- Stolk J, Hiltermann TJ, Dijkman JH, and Verhoeven AJ. Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. Am J Respir Cell Mol Biol 11: 95102, 1994.[Abstract]
- Suzuma I, Hata Y, Clermont A, Pokras F, Rook SL, Suyzuma K, Feener EP, and Aiello LP. Cyclic stretch and hypertension induce retinal expression of endothelial growth factor and vascular endothelial growth factor receptor-2: potential mechanisms for exacerbation of diabetic retinopathy by hypertension. Diabetes 50: 444454, 2001.[Abstract/Free Full Text]
- Thannickal M, Aldweib KDL, and Fanburg BL. Tyrosine phosphorylation regulates H2O2 production in lung fibroblasts stimulated by transforming growth factor
1. J Biol Chem 273: 2361123615, 1998.[Abstract/Free Full Text]
- Thannickal V, Day RM, Klinz SG, Bastien MC, Larios JM, and Fanburg BL. Ras-dependent and -independent regulation of reactive oxygen species by mitogenic growth factors and TGF-
1. FASEB J 14: 17411748, 2000.[Abstract/Free Full Text]
- Thannickal V and Fanburg BL. Activation of an H2O2-generating NADH oxidase in human lung fibroblasts by transforming growth factor
1. J Biol Chem 270: 3033430338, 1995.[Abstract/Free Full Text]
- Thannickal V, Hassoun PM, White AC, and Fanburg BL. Enhanced rate of H2O2 release from bovine pulmonary artery endothelial cells induced by TGF-
1. Am J Physiol Lung Cell Mol Physiol 265: L622L626, 1993.[Abstract/Free Full Text]
- Touyz R, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, and Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries. Circ Res 90: 12051213, 2002.[Abstract/Free Full Text]
- Wedgwood S, Dettman RW, and Black SM. ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 281: L1058L1067, 2001.[Abstract/Free Full Text]
- Zafari A, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, and Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension 32: 488495, 1998.[Abstract/Free Full Text]
- Zheng W, Seftor EA, Meininger CJ, Hendrix MJC, and Tomanek RJ. Mechanisms of coronary angiogenesis in response to stretch: role of VEGF and TGF-
. Am J Physiol Heart Circ Physiol 280: H909H917, 2001.[Abstract/Free Full Text]