© 2000 American Heart Association, Inc.
Vascular Biology |
Phenotypic Modulation by Fibronectin Enhances the Angiotensin II–Generating System in Cultured Vascular Smooth Muscle Cells
From the Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan.
Correspondence to Noboru Fukuda, MD, Second Department of Internal Medicine, Nihon University School of Medicine, Ooyaguchi-kami 30–1, Itabashi-ku, Tokyo 173-8610, Japan.
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Abstract—We previously demonstrated that homogeneous cultures of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats produce angiotensin II (Ang II) in response to increases in the levels of angiotensinogen, cathepsin D, and angiotensin-converting enzyme (ACE). The change of VSMCs from the contractile to the synthetic phenotype increased the amount of synthetic organelles, resulting in the production of proteases and growth factors. To evaluate the contribution of the synthetic phenotype to the generation of Ang II, we examined the effect of fibronectin (FN), which reportedly induces the synthetic phenotype, on the Ang II–generating system in VSMCs. Cultured VSMCs from Wistar-Kyoto rats were incubated with an active fragment of FN, Arg-Gly-Asp-Ser, for 24, 48, or 72 hours after synchronization of the cell cycle with 0.2% calf serum for 48 hours. Immunofluorescence and protein levels of
-smooth
muscle (SM) actin and expression of SM22 mRNA, apparent in the
contractile phenotype, were suppressed by FN, whereas
expression of matrix Gla mRNA and osteopontin mRNA and protein,
apparent in the synthetic phenotype, was increased. FN (1 to
1000 µg/mL) dose-dependently increased DNA synthesis in the VSMCs,
which was inhibited by the Ang II type 1 receptor
antagonist CV-11974. Ang II–like immunoreactivity as
determined by radioimmunoassay was significantly increased in
conditioned medium from the VSMCs. In addition, mRNA for the Ang
II–generating proteases cathepsin D and ACE was increased by FN.
Expression of transforming growth factor-ß1, platelet-derived
growth factor A-chain, and basic fibroblast growth factor mRNAs was
also increased by FN. These results indicate that the changes
accompanying the alteration to the synthetic phenotype in
homogeneous cultures of VSMCs increase expression of
proteases such as cathepsin D and ACE, which then produce Ang II, and
that these changes increase expression of growth factors that then
induce growth of VSMCs.
Key Words: angiotensin II • vascular smooth muscle • phenotype • rats • reverse transcription–polymerase chain reaction
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Introduction |
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Several lines of evidence have demonstrated the presence of a local tissue renin-angiotensin system (RAS) that is independent of the circulating RAS.1 2 The tissue RAS has been detected in brain, heart, kidney,1 and the vascular wall.2 The RAS in the vascular wall acts not only to regulate vessel tone and blood flow but also to induce vascular growth.3 The vascular wall contains renin, angiotensin-converting enzyme (ACE), angiotensinogen, angiotensin (Ang) I, and Ang II as components of the tissue RAS.1 2 Vessel walls are composed of endothelium, vascular smooth muscle, and extracellular matrix, and the smooth muscle contains renin, angiotensinogen, the Ang II receptors, and a small amount of ACE.4 Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) show an exaggerated growth compared with cells from Wistar-Kyoto rats (WKY).5 In a previous study, we detected Ang II–like immunoreactivity in homogeneous cultures of VSMCs from SHR, especially at early passages, but not in VSMCs from WKY.6 In addition, we demonstrated increasing levels of angiotensinogen, cathepsin D, and ACE mRNAs.6 However, we recently found Ang II–like immunoreactivity in late-passage VSMCs from WKY.
Freshly plated VSMCs do not proliferate, and they possess a high volume fraction of myofilaments, show few biosynthetic organelles, and retain their ability to contract in response to a vasoconstrictor. These properties are defined as the differentiated contractile phenotype. After several passages, VSMCs dedifferentiate and display the synthetic phenotype, which is characterized by a reduction in the volume fraction of myofilaments and an increase in synthetic organelles such as the Golgi, mitochondria, and endoplasmic reticulum, by which proteases, growth factors, and cytokines are extensively produced.7 8 It is possible, therefore, that the mechanism underlying Ang II generation in homogeneous cultures of VSMCs is associated with the change from the contractile to the synthetic phenotype.
Fibronectin (FN), a 500-kDa glycoprotein found in blood plasma and extracellular matrixes, is a dimeric molecule with binding affinities for heparin, fibrin, collagen, and cell surface components and participates in the regulation of cell migration, growth, and differentiation.9 FN has been known to promote the modulation of VSMCs from the contractile to the synthetic phenotype by interacting with integrins on the cell surface. This process is characterized by a structural and functional transformation of the cells, including a reorganization of the cytoskeleton, the formation of a large secretory apparatus, and the acquisition of a proliferative capacity.10
To investigate whether the change of VSMCs to the synthetic phenotype induces the generation of Ang II, we examined the effect of FN on the expression of phenotypic markers, Ang II–generating proteases, production of Ang II, and the expression of growth factors in VSMCs from WKY.
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Methods |
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Cell Culture
VSMCs were obtained by an explant method from aortas of 8-week-old male WKY/Izumo rats (SHR Corp, Funabashi, Chiba, Japan), as described previously.11 They were seeded and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% calf serum (Gibco Life Technologies), penicillin (100 U/mL), and streptomycin (100 µg/mL). The cells achieved confluence after 7 to 10 days, at which time they exhibited the "hill-and-valley" pattern characteristic of SMCs in culture. They were passaged by trypsinization with 0.05% trypsin (Gibco) in Ca2+- and Mg2+-free Dulbecco’s PBS and incubated in 80-cm2 tissue-culture flasks at a density of 105 cells/mL. No contamination with other cell types was apparent by light microscopy. Experiments were performed after 3 to 5 passages.
Quiescence was established by transferring the cells to 2-cm2 wells and incubating them for 24 hours in DMEM containing 10% calf serum and then for 48 to 72 hours in DMEM with 0.2% calf serum.
Determination of DNA Synthesis
[3H]thymidine incorporation into newly
synthesized DNA was assayed. In brief, quiescent VSMCs in
2-cm2 wells were incubated with or without 1 to
1000 µg/mL of the active fragment of FN (RGDS), Arg-Gly-Asp-Ser, in
the presence or absence of 10-6 mol/L CV-11974,
an Ang II type 1 receptor antagonist, for 24 hours in
serum-free DMEM. The medium was then changed to DMEM containing 0.5
µCi/mL [methyl-3H]thymidine
(specific activity, 20 Ci/mmol; NEN), and the cells were incubated for
an additional 2 hours. Each well was then washed with 1 mL of 150
mmol/L NaCl, and the cells were fixed with 1 mL of ethanol and acetic
acid (3:1, vol/vol) for 10 minutes. After the cells were washed with 1
mL of water, acid-insoluble material was precipitated with 1 mL of
ice-cold perchloric acid, and DNA was extracted into 1.5 mL of
perchloric acid by heating at 90°C for 20 minutes. The solubilized
DNA was transferred to a scintillation vial, and the associated
radioactivity was measured with a liquid scintillation
spectrometer.
Immunofluorescence for -SM
Actin
VSMCs were inoculated at 105
cells/cm2 in Laboratory Tek chamber slides (Nunc
Inc) into DMEM containing 10% calf serum. The culture medium was
changed to serum-free DMEM and the cells were incubated for 24 hours.
Then VSMCs were incubated with or without 1 mg/mL active fragment of FN
for 24, 48, or 72 hours. The culture medium was removed, and cells were
rinsed twice with PBS, fixed in methanol (-20°C, 5 minutes), and
dried for 2 hours at room temperature. The cells were incubated with
the first antibody (mouse monoclonal antibody specific for the -SM
actin isoform, DAKO A/S) diluted 1:100 in PBS for 1 hour and rinsed
with PBS for 30 minutes. Then the cells were incubated with the second
antibody (FITC-conjugated rabbit anti-mouse IgG, Zymed Laboratory)
diluted 1:20 in PBS for 60 minutes and rinsed with PBS for 30 minutes.
Photographs were taken on a Nikon Optiphot equipped for
epifluorescence with the use of Kodak Ektachrome 400 film.
Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Analysis for mRNAs of Phenotype Markers, Ang
II–Generating Proteases, and Growth Factors
Quiescent VSMCs at a density of 105
cells/cm2 in 2-cm2-well
dishes were incubated with or without 1 mg/mL active fragment of FN for
24, 48, or 72 hours. Then cells were washed with PBS and lysed in 800
µL of RNazol B (Biotex). Cell lysates were mixed with 80 µL of
chloroform, incubated at 4°C for 15 minutes, and centrifuged
at 12 000g for 15 minutes to extract total RNA. A portion
(300 µL) of each aqueous phase was mixed with an equal volume of
isopropanol, incubated at -20°C for 45 minutes, and
centrifuged at 12 000g for 15 minutes at 4°C to
precipitate the RNA. The RNA pellet was washed twice with 500 µL of
75% (vol/vol) ethanol by vortex mixing and
centrifugation at 7500g for 8 minutes at
4°C, dried, and then dissolved in 10 µL of a solution containing
10 mmol/L Tris-HCl (pH 8.0) and 1 mmol/L EDTA by incubation
for 15 minutes at 65°C. Each sample was then treated with 0.5 U of
DNase (Gibco) in 0.5 µL of DNase buffer (20 mmol/L Tris-HCl [pH
8.3], 50 mmol/L KCl, and 2.5 mmol/L
MgCl2) at room temperature for 45 minutes, after
which the DNase was inactivated by adding 0.5 µL of
20 mmol/L EDTA and heating at 98°C for 10 minutes.
RT-PCR was performed as described previously.12 In
brief, aliquots of RNA (1 µg/20 µL) were reverse-transcribed into
single-stranded cDNA by incubation for 10 minutes at 30°C, for 30
minutes at 42°C, and for 5 minutes at 99°C in a final volume of 20
µL containing 5 U of avian myeloblastoma virus reverse transcriptase
(Life Sciences), 10 mmol/L Tris-HCl (pH 8.3), 5 mmol/L
MgCl2, 50 mmol/L KCl, 1 mmol/L of each
deoxynucleotide triphosphate, and 2.5 µmol/L random
hexamers. The diluted cDNA products (5 µL) were then subjected to
PCR in a final volume of 25 µL containing 10 mmol/L Tris-HCl (pH
8.3), 50 mmol/L KCl, 4 mmol/L MgCl2,
0.625 U of Taq DNA polymerase (Takara), and 0.2
µmol/L each of upstream sense and downstream antisense primers. The
primers targeted to SM22 and matrix Gla genes were based on rat
SM22 and matrix Gla cDNA sequences.13 The primers
used for PCR for osteopontin,14 cathepsin
D,15 ACE,16 transforming growth factor
(TGF)-ß1,17 platelet-derived growth factor (PDGF)
A-chain,18 and basic fibroblast growth factor
(bFGF)19 were as previously described and are shown in
Table I (which appears online at
http://atvb.ahajournal.org). Rat ribosomal protein L19 mRNA was used as
an internal control.20 To confirm that no genomic DNA was
coamplified by PCR, control experiments of the RT-PCR without reverse
transcriptase but with every set of primers were performed, in which no
product was amplified. PCR was performed with a DNA thermal cycler
(Perkin-Elmer Cetus). For semiquantitative analysis of mRNA,
the kinetics of the PCR were monitored; the number of cycles at which
the PCR products became detectable on the gel was compared between
the different samples.21 Serial 10-fold dilutions of cDNA
(100, 10, and 1 ng) were amplified; the PCR products became
detectable at earlier cycles with increasing amounts of cDNA. Because
the amount of PCR product corresponding to each of the target mRNAs
increased in a linear manner from 20 to 35 cycles, PCR was performed
for 30 cycles according to the profiles shown in Table
II (which appears online at
http://atvb.ahajournal.org). PCR products were separated by
electrophoresis through 1.5% agarose gels and stained with ethidium
bromide.
Western Blot Analysis for -SM Actin and
Osteopontin Proteins
Quiescent VSMCs at a density of 105
cells/cm2 in 35-mm wells were incubated with or
without 1 mg/mL of the active fragment of FN for 24, 48, or 72 hours.
The cells were washed with PBS and lysed in lysis buffer (50
mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, 0.02%
NaN3, 100 µg/mL PMSF, 1 µg/mL aprotinin, and
1% Triton X). After centrifugation, the protein
concentration in the supernatant was determined by the method of Lowry
et al.22 Western blot analysis used 5 µg of
protein with 20 µL of sample buffer. The samples were boiled and
subjected to 10% polyacrylamide gel electrophoresis. The
proteins were transblotted to nitrocellulose membranes. After being
blocked with 100% Block Ace (Dainippon Pharmaceutical Co) at 4°C
overnight, the membranes were incubated with mouse monoclonal antibody
specific for -SM actin isoform (Dako), mouse monoclonal antibody
specific for osteopontin (American Research Products), or mouse
monoclonal antibody specific for -tubulin (Sigma Biosciences) as an
internal control diluted 1:200 (vol/vol) in TBST solution
(10 mmol/L Tris-HCl [pH 8.0], 150 mmol/L NaCl, and 0.05%
Tween 20) containing 15% Block Ace at room temperature for 3 hours.
After being washed with TBST for 10 minutes 2 times, the membranes were
incubated with goat anti-mouse IgG (Bio-Rad Laboratories) diluted
1:3000 (vol/vol) in TBST containing 15% Block Ace at room temperature
for 1 hour, washed with TBST for 10 minutes 3 times, and then stained
by addition of 10 mL of 0.1 mol/L Tris-HCl (pH 7.6) with 3 mg
diaminobenzidine HCl and 2 µL of 30%
H2O2.
Measurement of Ang II Level
VSMCs (106) from SHR and WKY were
inoculated in 2-cm2 wells with DMEM containing
10% calf serum and incubated for 24 hours. The cells were washed twice
with PBS and incubated with serum-free DMEM. The cells were incubated
with serum-free DMEM for 72, 48, 24, or 0 hours, and after each, the
cells were incubated with 1 mg/mL of the active fragment of FN for 0,
24, 48, or 72 hours, respectively. The culture medium was collected and
centrifuged at 600g for 10 minutes, and the
resulting supernatant (conditioned medium) was collected. The
conditioned medium was treated with 1 µmol/L each of aprotinin,
leupeptin, and pepstatin A as well as with 0.1 mmol/L PMSF and
stored at -80°C until analysis for Ang II by
radioimmunoassay.
Determination of angiotensin peptide levels in collected conditioned medium was made as described previously.23 Samples were applied to a Sep-Pak C18 cartridge (Waters Associates), and peptides were eluted with 3 mL of methanol/water/trifluoroacetic acid (80:19.9:0.1, vol/vol/vol). The eluate was dried in a vacuum centrifuge, and angiotensin peptides were separated by reversed-phase high-performance liquid chromatography. One milliliter of the collected conditioned medium was loaded on a Shodex column (OPD-50, Showa Denko), and peptides were eluted with an exponential gradient of acetonitrile from 20% to 50% (vol/vol) in 0.05% trifluoroacetic acid at a flow rate of 1 mL/min. Fractions (0.5 mL) were collected and dried in a vacuum centrifuge; the residues were redissolved in 0.1 mol/L Tris-HCl (pH 7.4) and subjected to radioimmunoassay for Ang II.
The antiserum to Ang II (Amersham) showed <1% cross-reactivity with Ang I and 100% cross-reactivity with Ang III (heptapeptide), Ang II(3–8) (hexapeptide), and Ang II(4–8) (pentapeptide). The sensitivity of detection of Ang II was 1 fmol per tube. The recoveries of Ang II were monitored by the addition of [3H]Ang II (Amersham) to conditioned media and cell extracts.
Compounds
The active fragment of FN was purchased from the Peptide
Institute. CV-11974 was purchased from Takeda Pharmaceutical Co,
Ltd.
Statistical Analysis
Values are shown as mean±SEM. The level of the significance of
differences between means was evaluated by Student’s t test
for unpaired data or by 2-way ANOVA followed by Duncan’s multiple
range test.
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Results |
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Effect of FN on VSMC Phenotype
To evaluate whether FN induces a change in VSMCs from WKY from the contractile to the synthetic phenotype, we examined the effect of FN over time on
-SM actin immunofluorescence;
on VSMC phenotypic markers such as SM22, expressed in the
contractile phenotype; and on matrix Gla and osteopontin,
expressed in the synthetic phenotype.
Immunofluorescence of -SM actin was decreased by
FN after 24 hours (Figure
I, which appears online at
http://atvb.ahajournal.org). The abundance of SM22 mRNA also
significantly decreased at 24 (P<0.05), 48, and 72
(P<0.01) hours, whereas the abundance of matrix Gla and
osteopontin mRNAs significantly (P<0.05) increased by 24
hours (Figure 1). Figure 2 shows Western blot analysis for
-SM actin and osteopontin proteins in VSMCs treated with FN. Western
blot analysis for -SM actin shows a major band with a
molecular weight of 40 kDa in VSMCs. Western blot analysis for
osteopontin shows 3 major bands with molecular weights of 80, 55, and
45 kDa in VSMCs. The variable composition of osteopontin protein is
consistent with a previous report.24 The
abundance of -SM actin protein also decreased at 24, 48, and 72
hours, whereas the abundance of osteopontin protein increased at 24,
48, and 72 hours. These results indicate that FN promotes the
alteration of VSMCs from the contractile to the synthetic
phenotype.
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Effect of FN on Growth of VSMCs
Figure 3 illustrates the effect of a
24-hour incubation with FN on DNA synthesis in VSMCs in the presence or
absence of 10-6 mol/L CV-11974, an Ang II type 1
receptor antagonist. Doses of 1 to 1000 µg/mL FN
significantly (P<0.05) increased DNA synthesis in VSMCs in
a dose-dependent manner. CV-11974 significantly (P<0.05)
inhibited DNA synthesis stimulated by the 1, 10, and 1000 µg/mL doses
of FN. These results indicate that FN stimulates growth of VSMCs
through the production of Ang II in these cells.
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P<0.05,
Effect of FN on Ang II–Generating Proteases
We next examined the effect of FN on the mRNA expression of
cathepsin D and ACE, proteases that contribute to Ang II generation in
VSMCs.6 The abundance of cathepsin D mRNA significantly
(P<0.05) increased at 24 hours and then decreased by 48 and
72 hours. The abundance of ACE mRNA significantly (P<0.05)
increased from 24 to 72 hours (Figure 4).
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Effect of FN on Ang II Production From VSMCs
Figure 5 shows the effect of FN on
Ang II–like immunoreactivity in conditioned medium from VSMCs. FN
significantly increased the amount of Ang II–like immunoreactivity in
the conditioned medium at 24 (P<0.01) and 48
(P<0.05) hours. Its peak level of increases in Ang II–like
immunoreactivity was seen 24 hours after the incubation, and then Ang
II–like immunoreactivity decreased by 72 hours.
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Effect of FN on Expression of Growth Factors
Figure 6 shows the time course of FN
treatment on the mRNA expression of TGF-ß1, PDGF A-chain, and bFGF.
The abundance of TGF-ß1 significantly increased at 24
(P<0.01) and 72 (P<0.05) hours, and PDGF
A-chain mRNAs significantly (P<0.05) increased at 24 hours.
The abundance of bFGF mRNA significantly (P<0.05) increased
from 24 to 72 hours.
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Discussion |
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Cultured VSMCs from SHR showed a higher specific growth rate, abnormal contact inhibition, and an accelerated entry into the S phase of the cell cycle compared with cells from WKY.1 2 We have demonstrated that SHR-derived VSMCs produce Ang II, which is associated with their exaggerated growth. VSMCs from SHR express increased mRNA levels for angiotensinogen and cathepsin D for conversion of angiotensinogen to Ang I and of ACE for conversion of Ang I to Ang II.6 These findings indicate that SHR-derived VSMCs possess the complete Ang II–generating system. The mechanism underlying this enhanced generation of Ang II in VSMCs from SHR is potentially due to the change from the contractile to the synthetic phenotype. To investigate whether the change to the synthetic phenotype contributes to Ang II production, we examined the effect of FN on the Ang II–generating system in homogeneous cultures of VSMCs from WKY.
Extracellular matrix components strongly influence the differentiated properties of VSMCs.25 26 27 FN mediates attachment and spreading and initiates a structural reorganization of the cells to the synthetic phenotype.25 In contrast, laminin and collagen type IV promote the contractile phenotype.25 27 FN was found to exert its effect via the cell attachment sequence Arg-Gly-Asp and by interaction with a cell surface receptor belonging to the integrin family of proteins.28 Hedin et al26 demonstrated that a substrate composed of the cell attachment sequence of FN (Arg-Gly-Asp-Ser) is sufficient to promote the transition of VSMCs from the contractile to the synthetic phenotype. Therefore, we treated VSMCs with this fragment to promote the cells from the contractile to the synthetic phenotype.
Using differential screening of a cDNA library derived from cultured
rat aortic VSMC RNA, Shanahan et al14 identified 7 genes
that are preferentially expressed by contractile VSMCs as the primary
culture: -SM actin, 
-SM actin, calponin, phospholamban,
tropoelastin, SM22, and CHIP28. In addition, 2 genes are
preferentially expressed in passaged cells that have downregulated
their contractile proteins: osteopontin and matrix Gla. SM22 is
expressed preferentially in differentiated VSMCs, whereas matrix Gla
and osteopontin are expressed strongly in dedifferentiated
VSMCs.13 We therefore used SM22 as a marker for the
contractile phenotype and matrix Gla and osteopontin as markers
for the synthetic phenotype in cultured VSMCs treated with FN.
FN decreased the immunofluorescence of -SM actin
and expression of SM22 mRNA, whereas it increased expression of
matrix Gla and osteopontin mRNAs, indicating that FN actually promotes
VSMCs from the contractile to the synthetic phenotype. In
addition, FN significantly increased Ang II levels in conditioned
medium from VSMCs and increased cathepsin D and ACE mRNA levels. VSMCs
displaying the synthetic phenotype are characterized by
increased numbers of synthetic organelles such as lysosomes,
Golgi, mitochondria, and endoplasmic reticulum, by which proteases are
extensively produced.7 8 These findings and our results
suggest that increases in cathepsin D and ACE are associated with the
increases in intracellular organelles that occur with the change to the
synthetic phenotype and that the increases in cathepsin D and
ACE generate Ang II.
FN significantly increased growth of VSMCs, which was inhibited by the Ang II type 1 receptor antagonist CV-11974. This result indicates that FN promotes growth of VSMCs through the production of Ang II in these cells. Ang II has been known to induce mRNA expression of TGF-ß1 mRNA, which then induces PDGF A-chain mRNA.29 30 Ang II also induces expression of bFGF mRNA.31 We recently demonstrated that increased expression of mRNA for TGF-ß1, PDGF A-chain, and bFGF is inhibited by an Ang II type 1 receptor antagonist in VSMCs from SHR, suggesting that endogenous production of Ang II is associated with the increased levels of these growth factors.32 In the current experiments, FN transiently increased Ang II levels in conditioned medium from VSMCs and expression of TGF-ß1 and PDGF A-chain mRNAs, findings that are consistent with increases in the expression of matrix Gla and osteopontin. These results indicate that FN changes VSMCs from the contractile to the synthetic phenotype to produce Ang II, which is associated with the increases in expression of TGF-ß1 and PDGF A-chain mRNAs. Hultgardh-Nilsson et al33 also investigated the effect of FN on the phenotypic change and the expression of growth factors by VSMCs in primary culture, and they found that FN transiently increased expression of osteopontin and PDGF A-chain mRNAs. That is consistent with our results showing transient increases in PDGF A-chain mRNA in FN-treated VSMCs. On the other hand, expression of bFGF mRNA was delayed relative to the increase in expression of phenotype markers, suggesting different mechanisms of induction of TGF-ß1 and PDGF A-chain mRNAs versus bFGF mRNA by Ang II.
The vascular RAS has been considered to be derived from the whole vessel, thus composed of the endothelium and VSMCs. However, our results indicate that VSMCs themselves possess the complete Ang II–generating system and are induced to change to the synthetic phenotype and that the generated Ang II is associated with growth of VSMCs.
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Acknowledgments |
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This work was financially supported in part by a grant-in aid for the High-Tech Research Center from the Japanese Ministry of Education, Science, Sports and Culture to Nihon University.
Received April 27, 1999; accepted March 21, 2000.
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