AJP - Lung Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L1459-L1466, 2007. First published February 23, 2007; doi:10.1152/ajplung.00143.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/L1459    most recent
00143.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, W.
Right arrow Articles by Raj, J. U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, W.
Right arrow Articles by Raj, J. U.

Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase

Weilin Zhou, Chiranjib Dasgupta, Sewite Negash, and J. Usha Raj

Division of Neonatology, Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, Torrance, California

Submitted 13 April 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic hypoxia triggers pulmonary vascular remodeling, which is associated with a modulation of the vascular smooth muscle cell (SMC) phenotype from a contractile, differentiated to a synthetic, dedifferentiated state. We previously reported that acute hypoxia represses cGMP-dependent protein kinase (PKG) expression in ovine fetal pulmonary venous SMCs (FPVSMCs). Therefore, we tested if altered expression of PKG could explain SMC phenotype modulation after exposure to hypoxia. Hypoxia-induced reduction in PKG protein expression strongly correlated with the repressed expression of SMC phenotype markers, myosin heavy chain (MHC), calponin, vimentin, {alpha}-smooth muscle actin ({alpha}SMA), and thrombospondin (TSP), indicating that hypoxic exposure of SMC induced phenotype modulation to dedifferentiated state, and PKG may be involved in SMC phenotype modulation. PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs significantly attenuated calponin, vimentin, and MHC expression, with no effect on {alpha}SMA and TSP. Treatment with 30 µM Drosophila Antennapedia (DT-3), a membrane-permeable peptide inhibitor of PKG, attenuated the expression of TSP, MHC, {alpha}SMA, vimentin, and calponin. The results from PKG siRNA and DT-3 studies indicate that hypoxia-induced reduction in protein expression was also similarly impacted by PKG inhibition. Overexpression of PKG in FPVSMCs by transfection with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) reversed the effect of hypoxia on the expression of SMC phenotype marker proteins. These results suggest that PKG could be one of the determinants for the expression of SMC phenotype marker proteins and may be involved in the maintenance of the differentiated phenotype in pulmonary vascular SMCs in hypoxia.

vein; small interfering RNA

IN BOTH ARTERIES and veins, the smooth muscle cell (SMC) coat is the site of active vasomotion. To be able to participate in regulation of vasomotor tone and vessel caliber, SMCs must maintain their contractile phenotype. Unlike terminally differentiated cells, SMCs are remarkably plastic and can undergo reversible changes in their phenotype in response to a variety of cues generated locally within the tissue (31). The high degree of plasticity of SMCs also predisposes the SMCs to respond to abnormal environmental cues, which may cause aberrant phenotypic switching and acquisition of characteristics that can contribute to the onset and/or progression of vascular disease (31).

SMC phenotype modulation has been implicated in a number of human diseases, namely, atherosclerosis, restenosis, and pulmonary hypertension. In systemic arteries, atherosclerosis and restenosis are the most widely acknowledged human diseases that are believed to involve SMC phenotypic modulation (19). At least part of the changes in the arterial wall structure is the result of medial vascular SMCs becoming highly proliferative and secretory. The transition of arterial SMCs from a contractile phenotype to a proliferative/secretory phenotype is an early event in systemic vascular changes associated with atherosclerosis and restenosis.

Relatively little information is available about vascular remodeling in veins, both systemic and pulmonary, and the involvement of SMC phenotype modulation in diseases of the pulmonary vasculature, for example, pulmonary hypertension. This disease has a complex etiology with poorly understood stimuli including chronic hypoxia, manifesting as aberrant vasoconstriction, dedifferentiation of vascular SMCs, and SMC proliferation. Change in vessel morphology is the prominent pathological feature in lungs subjected to chronic hypoxia. Although it is believed that it is mostly the arteries that undergo remodeling, there is a body of literature that clearly describes vascular remodeling in veins as well (reviewed in Ref. 17). This is particularly true in a number of species including sheep, rat, and human. Thus in patients with pulmonary hypertension, intimal and adventitial thickening of pulmonary veins <250 µm in diameter was observed in ~50% of the subjects studied. In adult sheep, after 20-h hypoxia, subsequent hypoxia caused veins to contract vigorously. Remodeling of veins was also corroborated by ultrastructural studies (17).

Phenotype modulation may also be demonstrated in vitro. SMCs undergo spontaneous changes in their behavioral phenotype during in vitro culture conditions (39). Such changes are associated with a transition from a contractile, differentiated to a synthetic, dedifferentiated phenotype, which has increased motility, proliferation and expression of extracellular matrix proteins. cGMP-dependent protein kinase (PKG) type 1 is critically involved in the endothelium-derived nitric oxide-cGMP signaling pathway that regulates a variety of cellular responses, the most well studied of which is the regulation of vasomotor tone (16, 17). Boerth et al. (5) and Lincoln et al. (23) first described a role of PKG in determination of aortic SMC phenotype and implicated its role in vascular disease. They demonstrated that constitutive expression of PKG1{alpha} restores the contractile phenotype in cultured aortic SMC, whereas inhibition of PKG reverses the phenotype (11).

Our laboratory previously reported that exposure of ovine fetal pulmonary veins and venous SMC to hypoxia resulted in downregulation of PKG activity and the expression of PKG protein and mRNA (16). This specific effect of hypoxia on expression of PKG was more prominent in pulmonary veins than in arteries (16). Therefore, in this study, we have investigated the effect of hypoxia on ovine fetal pulmonary venous SMC (FPVSMC) in culture. We also investigated the role of PKG in regulating the expression of SMC phenotype marker proteins by studying the effects of inhibition of PKG expression and of PKG overexpression in FPVSMCs. Our results indicate that the repression of SMC contractile marker proteins in hypoxia is related to PKG inhibition and that this effect of hypoxia can be reversed by PKG overexpression in cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. Primary cultures of vascular SMCs were prepared from pulmonary veins of three near-term ovine fetuses. All experiments were performed with cells at passages 4–6. The media used for cell growth and maintenance was DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 400 ng/ml amphotericin B, 160 U/ml of penicillin and streptomycin (Invitrogen). Cells were cultured in a humidified incubator with constant supply of 5% CO2 at 37°C.

Hypoxic treatment of cultured SMC. Cultured subconfluent FPVSMCs were subjected to 24, 48, and 72 h of normoxia or hypoxia at 37°C for at least five separate experiments. All studies were performed with growth media containing 0.5% FBS after starvation in 0.5% FBS for 16 h. Cells were made hypoxic by placing the cells in a special hypoxia chamber (C-chamber) into which a gas mixture of 0% O2, 5% CO2 and balance nitrogen was flowed in at a rate sufficient to keep oxygen concentration within the chamber at ~2%. The concentration of O2 within the C-chamber was constantly monitored and controlled with an oxygen controller (ProOxC, Biospherix). Under hypoxic conditions, the PO2 in the cell culture media was maintained around 30–40 Torr and was monitored by measuring the gas tensions in the culture media using a Radiometer blood gas analyzer. For normoxia experiments, cells were incubated in a humidified incubator with constant supply of 5% CO2 at 37°C.

At the end of each time point, plates were removed from hypoxia chamber or normoxia incubator. Total protein extracts were prepared from the cells, and expression of marker proteins was determined by immunoblotting.

Antibodies. All antibodies used for this study were purchased from Sigma-Aldrich except for anti-PKA, anti-PKG, which were obtained from Stressgen, and anti-actin were from Calbiochem. The anti-mouse IgG and anti-rabbit IgG linked to horseradish peroxidase were purchased from Amersham Biosciences.

Determination of protein concentration. Protein concentration was determined using Bio-Rad protein assay reagent with the help of a standard BSA plot.

Construction of PKG-targeted double-stranded small interfering RNA. The catalytic domain sequence of bovine PKG1{alpha} (GenBank acc. no. X16086), base pairs 999–2016 (Ref. 11), was used as the template to design the siRNA that targets PKG transcript. siRNAs were synthesized by Qiagen, and the double-stranded small interfering RNA (ds-siRNA) that we used to silence PKG was finally generated in our laboratory by annealing the two strands, namely, sense, r(CCU UCU UUA UCA UCA GUA A)dTdT, and antisense, r(UUA CUG AUG AUA AAG AAG G)dTdG.

Transfection of FPVSMCs with ds-siRNA to silence PKG. siRNA (75 ng) was transfected using a HiPerFect transfection kit (Qiagen) according to manufacturer's protocol after FPVSMCs were grown to 50–70% confluence in 12-well plates. To obtain a long-term silencing effect of siRNA, we transfected the cells with PKG siRNA sequentially twice within an interval of 72 h. No cell death was seen after transfection. In brief, 75 ng siRNA in ECR buffer (supplied with RNAifect reagents from Qiagen) was incubated at room temperature for 5 min. Then, HiPerFect reagent was added, and incubation continued for an additional 10 min. The HiPerFect-siRNA complex was next added directly to the cells and brought up to 1 ml final volume with culture media. After the cells became almost 95–100% confluent (within 48–72 h), the cells from each well were trypsinized, split into two wells, and transfected with same ds-siRNA using the same procedure as mentioned above. Cells were studied after reaching 95–100% confluence (within 72 h after the second transfection). In total, three wells were transfected. The first well was transfected with real PKG siRNA, the second well was transfected with a negative control siRNA (nonsilencing siRNA, which have no homology with any known mammalian gene; cat. no. 1022563, Qiagen), and the third well was mock transfected (i.e., without any ds-siRNA, but otherwise same). Whole cell lysate was prepared from each well in PKG assay buffer (described under PKG enzymatic activity assay) and total protein concentration was determined using Bradford reagent (Bio-Rad). PKG activity assay and Western blotting analysis were performed, and siRNA transfection efficiency was determined using fluorescently labeled siRNA (Qiagen). Western blots from at least five separate experiments were quantified by densitometry.

PKG enzyme activity assay. PKG activity was measured in the cell lysate derived from either real PKG or negative control siRNA transfection. PKG activity assay was performed following methods described by Gao et al. (16), with minor modifications necessary for cultured cells. Briefly, treated cells were sonicated on ice bath in a hypotonic buffer containing 50 mM Tris·HCl, pH 7.4, 10 mM EDTA, 2 mM DTT, 1 mM isobutylmethylxanthine, 100 µM nitro-L-arginine, and 10 µM indomethacin. The lysate was centrifuged in cold (4°C) at 12,000 g for 10 min, and clear supernatant was collected. Total protein content of the clear supernatant was measured, and 8 µg of total protein per lysate was used to determine PKG activity. PKG activity was finally expressed as picomole of 32P incorporation into PKG substrate, BPDEtide (Biomol), per minute per milligram of protein. PKG activity data were obtained from five separate experiments.

Determination of protein expression by immunoblotting. Immunoblotting was used to compare the expression of PKG, PKA, actin (including {alpha}-, beta-, and {gamma}- forms of actin), myosin heavy chain (MHC), vimentin, calponin, thrombospondin (TSP), and {alpha}-smooth muscle actin ({alpha}SMA) in the cells after the following treatments: 1) transfection with PKG siRNA, negative control siRNA, and mock transfection; 2) hypoxia and normoxia; 3) DT-3 and vehicle; and 4) transfection with PKG-green fluorescent fusion protein (PKG-GFP) or control vector. Protein samples were resolved through a 4–12% gradient NuPAGE Novex Bis-Tris mini-gel (Invitrogen) at 150 V and electrophoretically transferred to nitrocellulose membranes at 30 V for 60 min using a Xcell II blot module (Invitrogen). Membranes were blocked with blocking solution (10% nonfat powdered milk in TTBS: 20 mM Tris, pH 7.4, 137 mM NaCl, 0.1% Tween 20) and incubated at 4°C overnight with primary antibodies that were appropriately diluted in blocking solution. Membranes were then washed three times in TTBS, followed by incubation for 1 h at room temperature with 1:2,000–1:20,000 dilution of the secondary antibody. Membranes were again washed three times in TTBS, followed by incubation with SuperSignal West Pico Chemiluminescent Substrate System (Pierce) for signal generation, which was recorded on photosensitive film. Immunoblot images were digitized with a flatbed scanner (Epson). The band intensities were quantified using UN-SCAN-IT gel software v5.1 (Silk Scientific) and normalized with density of total actin. Differences among control and treated groups were expressed as percent of control (assigned 100%) and shown in bar diagrams with means ± SE, wherever applicable. Western blots from at least five separate experiments were quantified by densitometry.

Treatment of FPVSMCs with PKG inhibitor DT-3. Approximately 70% confluent FPVSMCs were treated with the peptide PKG inhibitor DT-3 (Drosophila Antennapedia homeo-domain; RQIKIWFQNRRMKWKK-LRK(5)H, EMD) at 3 x 10–5 M concentration for 48 h in the growth media. Fresh inhibitor was applied every day. On completion of treatment, cell lysates were prepared, and effect of DT-3 on protein expression was analyzed by immunoblotting. Western blots from at least five separate experiments were quantified by densitometry.

Overexpression of PKG in FPVSMCs. A plasmid encoding a full-length PKG1{alpha} tagged with GFP (PKG-GFP) (8) was kindly provided by Dr. Darren D. Browning, Department of Pharmacology, University of Illinois at Chicago. Transient transfection was performed using Lipofectamine (Invitrogen). Briefly, cells were seeded at a density of ~6.3 x 104/cm2 in DMEM with 10% FBS and allowed to attach overnight. Transfection was performed 1 day after seeding using 1 µg of DNA and 2 µl of Lipofectamine per 105 cells following the manufacturer's protocol. FPVSMCs were transfected with PKG-GFP or vector alone (pEGFP-N1 vector, Clontech) as a control. Cells were trypsinized and seeded in six-well plates the day after transfection. One day after seeding, cells were treated with hypoxia for 1 day. Western blots from at least four separate experiments were quantified by densitometry.

Data analysis. SigmaStat v3.1 software was used for data analysis. Unpaired t-tests were used to compare differences in density of Western blots, and a probability value under 0.05 was considered significant. If the data did not pass the normality test, Mann-Whitney rank sum tests were used as an alternate, as recommended by the software SigmaStat. Relationship between the protein expression level of PKG and the SMC marker proteins was determined by the Pearson product-moment correlation analysis, and P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia downregulates the expression of PKG and SMC marker proteins. The effect of hypoxia on expression of PKG and SMC phenotype marker proteins was studied over a period of 24–72 h. After 24, 48, and 72 h of hypoxia, the expression of PKG was downregulated to ~40%, ~65%, and ~55% of the level in normoxia, respectively (Fig. 1, A and B). The differences in expression of PKG at each time point between hypoxia and normoxia were statistically significant (P < 0.05).


Figure 1
View larger version (47K):
[in this window]
[in a new window]

  		Fig. 1. Expression of PKG in fetal pulmonary venous smooth muscle cells (FPVSMCs) is decreased after hypoxia (Western blot). Cells were incubated in normoxia (N) or hypoxia (H or Hyp) for the indicated time periods (24, 48, and 72 h). A: representative Western blots. B: quantification of Western blots by densitometry. *Significantly different from respective normoxia controls (expressed as 100%; P < 0.05).

 
After exposure of FPVSMCs to hypoxia, the expressions of SMC phenotype marker proteins, MHC, {alpha}SMA, calponin, vimentin, and TSP (Fig. 2) were significantly decreased (P < 0.05) at each time point, except there was no statistically significant difference between the expression of {alpha}SMA in hypoxia and normoxia at 24 h (P = 0.710).


Figure 2
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 2. Expression of smooth muscle cell (SMC) marker proteins in FPVSMCs is downregulated after hypoxia (Western blot). Cells were incubated in normoxia (N) or hypoxia (H or Hyp) for the indicated time periods (24, 48, and 72 h). A: representative Western blots. B: quantification of Western blots by densitometry. *Significantly different from respective normoxia controls (P < 0.05). TSP, thrombospondin; MHC, myosin heavy chain; {alpha}SMA, {alpha}-smooth muscle actin.

 
To determine if the downregulation of PKG correlates with the downregulation of SMC phenotype marker proteins, Pearson product-moment correlation analysis was performed with the Western blot density data after hypoxia treatment. The analysis (Table 1) showed that the downregulation of TSP, calponin, vimentin, and MHC proteins all positively correlated with PKG downregulation after hypoxia treatment at each of the different time points (P < 0.05), except for the fact that the change in protein expression level of vimentin did not significantly correlate with downregulation of PKG after hypoxia for 48 h (P > 0.05). The overall positive correlation indicates that the downregulation of SMC phenotype marker protein expression (TSP, vimentin, calponin, and MHC) is related to the downregulation of PKG expression in hypoxia. Although after hypoxia for 48 h and 72 h, the expression of {alpha}SMA was decreased, and the change did not statistically correlate with changes in PKG expression.


View this table:
[in this window]
[in a new window]

  		Table 1. Correlation of expression levels of PKG and SMC marker proteins after hypoxia

 
The above results indicate that FPVSMCs change from a differentiated to a dedifferentiated phenotype in hypoxia and that the downregulation of PKG may play an important role in modulating cell phenotype after hypoxic exposure.

Inhibition of PKG downregulates the expression of SMC marker proteins. To further understand the role of PKG in modulating cell phenotype after hypoxia, we inhibited PKG by PKG siRNA or a pharmacological inhibitor to mimic the effect of hypoxia on PKG and then determined the expression of SMC phenotype marker proteins.

PKG-specific siRNA was designed and transfected to FPVSMCs. Our results (Fig. 3) showed that in FPVSMCs transfected with PKG siRNA, protein expression level of PKG was decreased to ~25%, and PKG activity was decreased to ~40% of the level in FPVSMCs transfected with negative control siRNA. The decreases in PKG protein expression and activity were statistically significant (P < 0.05). We did not find any significant silencing of PKA-specific protein expression. Thus the PKG siRNA used in this study was PKG specific. The expression of SMC phenotype marker proteins (MHC, vimentin, and calponin) was significantly downregulated (P < 0.05) in FPVSMCs transfected with PKG siRNA (Fig. 4). Although the expression levels of TSP and {alpha}SMA were slightly decreased, the changes were not statistically significant (P > 0.05).


Figure 3
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 3. Protein expression level and activity of PKG are suppressed by PKG-specific small interfering RNA (siRNA) transfection in FPVSMCs. Cells were transfected with PKG-specific siRNA and nonsilencing negative control siRNA (Control) and studied after 6 days of transfection. Mock transfection was also performed. A and B: representative Western blots and quantification of Western blots by densitometry. *Significantly different from respective control (P < 0.05). C: PKG activity assay. *Significantly different from control (P < 0.05).

 

Figure 4
View larger version (34K):
[in this window]
[in a new window]

  		Fig. 4. Expression of SMC marker proteins in FPVSMCs is downregulated after PKG siRNA transfection. PKG-specific siRNA and nonsilencing negative control siRNA (Control) were transfected into FPVSMCs. A: representative Western blots. B: quantification of Western blots by densitometry. *Significantly different from respective control (P < 0.05).

 
Next, we used DT-3, a highly specific membrane-permeable peptide inhibitor of PKG1{alpha} (13), to determine if the effect of hypoxia on SMC phenotype could be mimicked by inhibition of PKG activity. Figure 5 showed that treatment of FPVSMCs with 30 µM DT-3 for 48 h resulted in significant downregulation of expression of all the SMC marker proteins that we had studied in hypoxia (P < 0.05).


Figure 5
View larger version (39K):
[in this window]
[in a new window]

  		Fig. 5. Expression of SMC marker proteins in FPVSMCs is downregulated after DT-3 treatment. Cells were treated with or without the PKG inhibitor peptide, DT-3, at a concentration of 30 µM for 48 h. A: representative Western blots. B: quantification of Western blots by densitometry. *Significantly different from respective control (P < 0.05).

 
PKG overexpression in FPVSMCs reverses the effect of hypoxia on SMC marker proteins. To further define the role of PKG in hypoxia-induced downregulation of SMC marker proteins, we overexpressed PKG by transfecting with PKG-GFP (expressing a full-length PKG1{alpha} tagged with GFP) in FPVSMCs and then incubated the cells in hypoxia for 24 h. Figure 6A showed that the expression of PKG in PKG-GFP transfectants was much higher than in vector-only transfectants. This confirms that PKG overexpression in FPVSMCs was successful. Figure 6, B and C, showed that in vector-transfected cells, the SMC marker protein expression profile after hypoxia was similar to nontransfected FPVSMCs treated with hypoxia, i.e., there was downregulation of most SMC marker proteins including TSP, MHC, vimentin, and calponin. However, in PKG-GFP transfectants after hypoxic exposure, the SMC marker protein expression profile was significantly different from that in vector-only transfectants (P < 0.05) and demonstrated that the marker proteins were not downregulated, which indicates that overexpression of PKG1{alpha} in FPVSMCs can reverse the downregulation of SMC marker protein expression after hypoxia.


Figure 6
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 6. PKG overexpression in FPVSMCs reverses hypoxia-induced SMC phenotype modulation. Cells were transiently transfected with a full-length PKG construct tagged with green fluorescent fusion protein (PKG-GFP) or control vector (pEGFP-N1). Transfected cells were replated and treated with hypoxia (Hyp) or normoxia (Nor) for 24 h. A: representative Western blots showing PKG expression. B: representative Western blots showing SMC marker protein expression. C: quantification of Western blots by densitometry. *Significantly different from vector-transfectant after hypoxia (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study to demonstrate the role of PKG in hypoxia-induced SMC phenotype modulation. We selected ovine FPVSMC as our model system because our laboratory (16) has previously demonstrated that PKG is downregulated in acute hypoxia in this cell type. This system thus provides us with a tool to test the hypothesis that altered PKG protein expression during hypoxia may also induce SMC phenotype modulation.

SMC phenotype modulation plays an integral role in pulmonary hypertension, atherosclerosis, and leiomyogenic tumorigenicity. During this process, vascular SMCs undergo a transition in phenotype from a contractile, differentiated to a synthetic, dedifferentiated state (reviewed in Ref. 24). A variety of SMC-selective or -specific genes and gene products have been identified that serve as useful markers of the relative state of differentiation-maturation of the SMC. {alpha}SMA is an excellent SMC differentiation marker because it is the first known protein expressed in differentiation of the SMC during development and is required for the high force development properties of fully differentiated SMCs (31). MHC is also a good and reliable SMC differentiation marker since it seems to be highly restricted to SMCs, whereas other proteins including calponin and {alpha}SMA are also expressed in fibroblasts (27). Calponin, a calcium regulatory protein, is also highly expressed in SMCs (31). The three proteins mentioned above have been used in this study as markers of a differentiated phenotype. Our data were consistent with results of other published studies on SMC phenotype modulation in that after hypoxia treatment, FPVSMCs expressed less SMC contractile marker proteins, which indicated that hypoxia induced phenotype modulation of FPVSMCs from a differentiated to a dedifferentiated state. Increased expression of vimentin in the vascular SMCs has been associated with vascular injury and used as a marker of SMC synthetic, dedifferentiated phenotype (2). Both vimentin and desmin are cytoskeletal proteins of the intermediate filament, but vimentin is only expressed in vascular SMC, whereas desmin is mostly expressed in visceral SMCs (14). Since desmin is believed to be one of the SMC contractile markers (31), vimentin was used as a marker for the contractile, differentiated SMC phenotype in this study. The downregulation of vimentin by hypoxia and by PKG inhibition seen in this study is also evidence that hypoxia did induce phenotype modulation of FPVSMCs from a differentiated to a dedifferentiated state. Similar to our results, Scheurer et al. (33) reported that in human umbilical cord vein endothelial cells, hypoxia-induced downregulation of vimentin in the cytoplasmic fraction was around 40%, but vimentin remained unchanged in whole cell lysate after hypoxia.

TSP, an extracellular matrix protein, is usually expressed more in synthetic SMC and used as a marker of SMC dedifferentiated phenotype (23). Our data show that hypoxia repressed the expression of TSP, which appears as if hypoxia modulates FPVSMCs to a less synthetic state. The seeming discrepancy of our data with other published data is probably because of the following reasons. First, it has previously been noted that vascular SMCs can exhibit different stable phenotypes in cell culture depending, for example, on their origin or the age of the animal from which they were isolated (3, 4, 20, 29, 30). The published SMC phenotype modulation studies are mostly in systemic arterial SMC, whereas our study was conducted in pulmonary venous SMC. Second, previous studies have shown that vascular SMC behaves differently during the development process from a fetus, neonate to an adult. There is a period during early postnatal vascular development when cells that are already committed to a vascular SMC lineage express high levels of genes coding for contractile proteins and high levels of genes for matrix proteins (34). In this study, we isolated SMC from ovine fetal pulmonary veins cultured in normoxic conditions and performed the experiments with passages 4–6 cells. Since normoxic culture conditions mimic the conditions after birth, the FPVSMCs used in this study may express more contractile proteins and synthetic proteins similar to neonatal SMC. Once treated with hypoxia condition at the same oxygen tension as in the fetal state, the cells may reverse to the fetal state and express less contractile and synthetic proteins. Third, hypoxia is a different stimulus from other vascular injury stimuli that the other SMC phenotypic studies have employed. The period of hypoxia treatment may also determine the level of protein expression. In in vivo hypoxia studies, mRNA expression level of TSP-1 in mouse posterior eyecups was downregulated after 24 h of hypoxia and remains downregulated through 48 h. Expression of TSP-1 was increased 1.6-fold at 96 h after hypoxia (9). Inhibition of expression of TSP in hypoxia is, however, consistent with the fact that TSP is an inhibitor of angiogenesis, which is known to be augmented by hypoxia (21). In this study, the expression of TSP was modulated both by inhibition of PKG and by overexpression of PKG, which indicates that PKG is the modulator of TSP expression in FPVSMCs.

Boerth et al. (5) published that overexpression of PKG transformed the "synthetic" rat aortic SMC (RASMC) to a more contractile-like morphology. Consistent with the morphology, overexpression of PKG resulted in an upregulation of contractile phenotype marker proteins, most noteworthy of which were MHC, calponin, and {alpha}SMA. In addition, overexpression of PKG in RASMC, but not the empty vector, reestablished contractile function monitored by the silicone polymer wrinkle assay (7). In contrast to the effect of PKG on contractile protein markers, RASMC transfected with PKG construct demonstrated reduced levels of TSP-1 protein compared with control-transfected RASMC (11). Conversely, the cell-permeable PKG specific inhibitor DT-2 transformed the contractile SMC to a synthetic state along with downregulation of proteins of contractile SMC, namely, MHC, calponin, smoothelin, and caldesmon (12). Taken together, these results indicate that PKG expression is required to maintain the contractile properties of cultured SMC and that the level of expression of some key protein markers of SMC could indicate the phenotype of the cell. Data from our studies, in which we inhibited PKG activity and overexpressed PKG in FPVSMCs, demonstrate that attenuation of MHC, calponin, and {alpha}SMA expression involves a pathway, which includes PKG as a key determinant factor. It is possible that this pathway may be inhibited in hypoxia.

SMC phenotype not only determines the expression pattern of marker proteins, but also determines the proliferative ability of SMC. Overexpression of PKG in RASMC, which promotes contractile phenotype, attenuated cell proliferation (37). PKG expression was transiently reduced in the population of vascular SMCs that were actively proliferating in response to balloon catheter injury in the swine coronary artery (1). In general, proliferation and PKG expression are reciprocally related in arterial SMCs. Contrary to these reports, Wolfsgruber et al. (38) reported that activation of endogenous PKG in mouse primary aortic SMCs resulted in cells with increased levels of proliferation. Therefore, the physiological role of PKG in regulating proliferation or phenotype might be different in different cell types and cell passages. Similar to arterial injury, hypoxia also promotes cell proliferation of cultured arterial SMC isolated from rat and rabbit (26, 39, 40). Quite a contrasting picture emerged from studies on human bladder SMCs, where hypoxia inhibited cell proliferation (15). Thus hypoxia may also impose opposite effects on SMCs derived from different tissue types.

It is known that the expression of SMC marker protein is regulated at the transcription level when SMC phenotype switches from a differentiated to a dedifferentiated state or vice versa (35, 36). Serum response factor (SRF), a transcription factor, is a regulator of SMC-specific gene expression and differentiation with higher expression in SMCs (6, 10). SRF is a nuclear protein that binds to its DNA target sequence (the "CArG box," CC[A/T]6GG) in the regulatory regions of most SMC contractile marker protein-encoding genes and so activates their transcriptional expression (31). Hypoxia is known to modulate SRF (22), and it is possible that hypoxia-induced modulation of PKG might regulate the availability of SRF and thus modulate the gene expression of SMC phenotype marker proteins. Activation of RhoA stimulates SRF-dependent transcription (25, 28). PKG signaling can inhibit RhoA functions such as Ca2+ sensitization of vascular SMC contractility and RhoA-dependent actin polymerization (32) and eventually inhibits SRF-dependent transcription indirectly by interfering with RhoA signaling (18). However, a second layer of complexity is imposed by the fact that, in different cells, PKG mediates opposite effects. Association of PKG with cell-specific effector molecules could dictate this process. Thus further studies will be necessary to determine the relationship between PKG and SRF and their involvement in SMC-specific gene expression in chronic hypoxia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-75187 and HL-59435 (to J. Usha Raj).


   ACKNOWLEDGMENTS
 
We thank Drs. Y. Gao and B. Ibe for helpful discussions. We also thank Nik Phou for excellent secretarial assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. Zhou, Division of Neonatology, Harbor-UCLA Medical Center, Los Angeles Biomedical Research Institute at Harbor-UCLA, 1124 West Carson St., Torrance, CA 90502 (e-mail: wzhou{at}labiomed.org)

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
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Anderson PG, Boerth NJ, Liu M, McNamara DB, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase expression in coronary arterial smooth muscle in response to balloon catheter injury. Arterioscler Thromb Vasc Biol 20: 2192–2197, 2000.[Abstract/Free Full Text]
  2. Asada H, Paszkowiak J, Teso D, Alvi K, Thorisson A, Frattini JC, Kudo FA, Sumpio BE, Dardik A. Sustained orbital shear stress stimulates smooth muscle cell proliferation via the extracellular signal-regulated protein kinase 1/2 pathway. J Vasc Surg 42: 772–780, 2005.[CrossRef][Web of Science][Medline]
  3. Bochaton-Piallat ML, Gabbiani F, Ropraz P, Gabbiani G. Age influences the replicative activity and the differentiation features of cultured rat aortic smooth muscle cell populations and clones. Arterioscler Thromb 13: 1449–1455, 1993.[Abstract/Free Full Text]
  4. Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol 16: 815–820, 1996.[Abstract/Free Full Text]
  5. Boerth NJ, Dey NB, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. J Vasc Res 34: 245–259, 1997.[Web of Science][Medline]
  6. Borrione AC, Zanellato AM, Giuriato L, Scannapieco G, Pauletto P, Sartore S. Nonmuscle and smooth muscle myosin isoforms in bovine endothelial cells. Exp Cell Res 190: 1–10, 1990.[CrossRef][Web of Science][Medline]
  7. Brophy CM, Woodrum DA, Pollock J, Dickinson M, Komalavilas P, Cornwell TL, Lincoln TM. cGMP-dependent protein kinase expression restores contractile function in cultured vascular smooth muscle cells. J Vasc Res 39: 95–103, 2002.[CrossRef][Web of Science][Medline]
  8. Browning DD, Mc Shane M, Marty C, Ye RD. Functional analysis of type 1alpha cGMP-dependent protein kinase using green fluorescent fusion proteins. J Biol Chem 276: 13039–13048, 2001.[Abstract/Free Full Text]
  9. Chan CK, Pham LN, Zhou J, Spee C, Ryan SJ, Hinton DR. Differential expression of pro- and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization. Lab Invest 85: 721–733, 2005.[CrossRef][Web of Science][Medline]
  10. Croissant JD, Kim JH, Eichele G, Goering L, Lough J, Prywes R, Schwartz RJ. Avian serum response factor expression restricted primarily to muscle cell lineages is required for alpha-actin gene transcription. Dev Biol 177: 250–264, 1996.[CrossRef][Web of Science][Medline]
  11. Dey NB, Boerth NJ, Murphy-Ullrich JE, Chang PL, Prince CW, Lincoln TM. Cyclic GMP-dependent protein kinase inhibits osteopontin and thrombospondin production in rat aortic smooth muscle cells. Circ Res 82: 139–146, 1998.[Abstract/Free Full Text]
  12. Dey NB, Foley KF, Lincoln TM, Dostmann WR. Inhibition of cGMP-dependent protein kinase reverses phenotypic modulation of vascular smooth muscle cells. J Cardiovasc Pharmacol 45: 404–413, 2005.[CrossRef][Web of Science][Medline]
  13. Dostmann WR, Taylor MS, Nickl CK, Brayden JE, Frank R, Tegge WJ. Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation. Proc Natl Acad Sci USA 97: 14772–14777, 2000.[Abstract/Free Full Text]
  14. Gabbiani G, Schmid E, Winter S, Chaponnier C, de Ckhastonay C, Vandekerckhove J, Weber K, Franke WW. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci USA 78: 298–302, 1981.[Abstract/Free Full Text]
  15. Galvin DJ, Watson RW, O'Neill A, Coffey RN, Taylor C, Gillespie JI, Fitzpatrick JM. Hypoxia inhibits human bladder smooth muscle cell proliferation: a potential mechanism of bladder dysfunction. Neurourol Urodyn 23: 342–348, 2004.[CrossRef][Web of Science][Medline]
  16. Gao Y, Dhanakoti S, Trevino EM, Sander FC, Portugal AM, Raj JU. Effect of oxygen on cyclic GMP-dependent protein kinase-mediated relaxation in ovine fetal pulmonary arteries and veins. Am J Physiol Lung Cell Mol Physiol 285: L611–L618, 2003.[Abstract/Free Full Text]
  17. Gao Y, Raj JU. Role of veins in regulation of pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 288: L213–L226, 2005.[Abstract/Free Full Text]
  18. Gudi T, Chen JC, Casteel DE, Seasholtz TM, Boss GR, Pilz RB. cGMP-dependent protein kinase inhibits serum-response element-dependent transcription by inhibiting rho activation and functions. J Biol Chem 277: 37382–37393, 2002.[Abstract/Free Full Text]
  19. Hao H, Gabbiani G, Bochaton-Piallat ML. Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development. Arterioscler Thromb Vasc Biol 23: 1510–1520, 2003.[Abstract/Free Full Text]
  20. Holifield B, Helgason T, Jemelka S, Taylor A, Navran S, Allen J, Seidel C. Differentiated vascular myocytes: are they involved in neointimal formation? J Clin Invest 97: 814–825, 1996.[Web of Science][Medline]
  21. Howell K, Preston RJ, McLoughlin P. Chronic hypoxia causes angiogenesis in addition to remodelling in the adult rat pulmonary circulation. J Physiol 547: 133–145, 2003.[Abstract/Free Full Text]
  22. Kwapiszewska G, Wilhelm J, Wolff S, Laumanns I, Koenig IR, Ziegler A, Seeger W, Bohle RM, Weissmann N, Fink L. Expression profiling of laser-microdissected intrapulmonary arteries in hypoxia-induced pulmonary hypertension. Respir Res 6: 109–124, 2005.[CrossRef][Medline]
  23. Lincoln TM, Dey NB, Boerth NJ, Cornwell TL, Soff GA. Nitric oxide–cyclic GMP pathway regulates vascular smooth muscle cell phenotypic modulation: implications in vascular diseases. Acta Physiol Scand 164: 507–515, 1998.[Web of Science][Medline]
  24. Lincoln TM, Wu X, Sellak H, Dey N, Choi CS. Regulation of vascular smooth muscle cell phenotype by cyclic GMP and cyclic GMP-dependent protein kinase. Front Biosci 11: 356–367, 2006.[Web of Science][Medline]
  25. Liu HW, Halayko AJ, Fernandes DJ, Harmon GS, McCauley JA, Kocieniewski P, McConville J, Fu Y, Forsythe SM, Kogut P, Bellam S, Dowell M, Churchill J, Lesso H, Kassiri K, Mitchell RW, Hershenson MB, Camoretti-Mercado B, Solway J. The RhoA/Rho kinase pathway regulates nuclear localization of serum response factor. Am J Respir Cell Mol Biol 29: 39–47, 2003.[Abstract/Free Full Text]
  26. Lu SY, Wang DS, Zhu MZ, Zhang QH, Hu YZ, Pei JM. Inhibition of hypoxia-induced proliferation and collagen synthesis by vasonatrin peptide in cultured rat pulmonary artery smooth muscle cells. Life Sci 77: 28–38, 2005.[CrossRef][Web of Science][Medline]
  27. Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res 75: 803–812, 1994.[Abstract/Free Full Text]
  28. Miralles F, Posern G, Zaromytidou AI, Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113: 329–342, 2003.[CrossRef][Web of Science][Medline]
  29. Neuville P, Geinoz A, Benzonana G, Redard M, Gabbiani F, Ropraz P, Gabbiani G. Cellular retinol-binding protein-1 is expressed by distinct subsets of rat arterial smooth muscle cells in vitro and in vivo. Am J Pathol 150: 509–521, 1997.[Abstract]
  30. Orlandi A, Ehrlich HP, Ropraz P, Spagnoli LG, Gabbiani G. Rat aortic smooth muscle cells isolated from different layers and at different times after endothelial denudation show distinct biological features in vitro. Arterioscler Thromb 14: 982–989, 1994.[Abstract/Free Full Text]
  31. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 84: 767–801, 2004.[Abstract/Free Full Text]
  32. Sauzeau V, Le Jeune H, Cario-Toumaniantz C, Smolenski A, Lohmann SM, Bertoglio J, Chardin P, Pacaud P, Loirand G. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem 275: 21722–21729, 2000.[Abstract/Free Full Text]
  33. Scheurer SB, Rybak JN, Rosli C, Neri D, Elia G. Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells: a transcriptomic and proteomic study. Proteomics 4: 1737–1760, 2004.[CrossRef][Web of Science][Medline]
  34. Shanahan CM, Weissberg PL. Smooth muscle cell heterogeneity: patterns of gene expression in vascular smooth muscle cells in vitro and in vivo. Arterioscler Thromb Vasc Biol 18: 333–338, 1998.[Abstract/Free Full Text]
  35. Sobue K, Hayashi K, Nishida W. Expressional regulation of smooth muscle cell-specific genes in association with phenotypic modulation. Mol Cell Biochem 190: 105–118, 1999.[CrossRef][Web of Science][Medline]
  36. Sobue K, Hayashi K, Nishida W. Molecular mechanism of phenotypic modulation of smooth muscle cells. Horm Res 50, Suppl 2: 15–24, 1998.[CrossRef][Web of Science][Medline]
  37. Stegemann JP, Dey NB, Lincoln TM, Nerem RM. Genetic modification of smooth muscle cells to control phenotype and function in vascular tissue engineering. Tissue Eng 10: 189–199, 2004.[CrossRef][Web of Science][Medline]
  38. Wolfsgruber W, Feil S, Brummer S, Kuppinger O, Hofmann F, Feil R. A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells. Proc Natl Acad Sci USA 100: 13519–13524, 2003.[Abstract/Free Full Text]
  39. Worth NF, Rolfe BE, Song J, Campbell GR. Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins. Cell Motil Cytoskeleton 49: 130–145, 2001.[CrossRef][Web of Science][Medline]
  40. Yu TZ, Ma CT. Effects of angiotensin-converting enzyme and angiotensin II on hypoxia-induced proliferation of cultured intra-pulmonary artery smooth muscle cells. Acta Pharmacol Sin 21: 381–384, 2000.[Web of Science][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Negash, S. R. Narasimhan, W. Zhou, J. Liu, F. L. Wei, J. Tian, and J. U. Raj
Role of cGMP-dependent protein kinase in regulation of pulmonary vascular smooth muscle cell adhesion and migration: effect of hypoxia
Am J Physiol Heart Circ Physiol, July 1, 2009; 297(1): H304 - H312.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
W. Zhou, S. Negash, J. Liu, and J. U. Raj
Modulation of pulmonary vascular smooth muscle cell phenotype in hypoxia: role of cGMP-dependent protein kinase and myocardin
Am J Physiol Lung Cell Mol Physiol, May 1, 2009; 296(5): L780 - L789.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/L1459    most recent
00143.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (6)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhou, W.
Right arrow Articles by Raj, J. U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhou, W.
Right arrow Articles by Raj, J. U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.