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. 2013 Aug 22;46(5):563–575. doi: 10.1111/cpr.12052

RhoA–Rho kinase and platelet‐activating factor stimulation of ovine foetal pulmonary vascular smooth muscle cell proliferation

L S Renteria 1, M Austin 1, M Lazaro 1, M A Andrews 1, J Lustina 1, J U Raj 2, B O Ibe 1,✉
PMCID: PMC3788060  NIHMSID: NIHMS500396  PMID: 24033386

Abstract

Objectives

Platelet‐activating factor (PAF) is produced by pulmonary vascular smooth muscle cells (PVSMC). We studied effects of Rho kinase on PAF stimulation of PVSMC proliferation in an attempt to understand the role of RhoA/Rho kinase on PAF‐induced ovine foetal pulmonary vascular remodelling. Our hypothesis is that PAF acts through Rho kinase, as one of its downstream signals, to induce arterial (SMC‐PA) and venous (SMC‐PV) cell proliferation in the hypoxic lung environment of the foetus, in utero.

Materials and methods

Rho kinase and MAPK effects on PAF receptor (PAFR)‐mediated cell population expansion, and PAFR expression, were studied by DNA synthesis, western blot analysis and immunocytochemistry. Effects of constructs T19N and G14V on PAF‐induced cell proliferation were also investigated.

Results

Hypoxia increased PVSMC proliferation and Rho kinase inhibitors, Y‐27632 and Fasudil (HA‐1077) as well as MAPK inhibitors PD 98059 and SB 203580 attenuated PAF stimulation of cell proliferation. RhoA T19N and G14V stimulated cell proliferation, but co‐incubation with PAF did not affect proliferative effects of the constructs. PAFR protein expression was significantly downregulated in both cell types by both Y‐27632 and HA‐1077, with comparable profiles. Also, cells treated with Y‐27632 had less PAF receptor fluorescence with significant disruption of cell morphology.

Conclusions

Our results show that Rho kinase non‐specifically modulated PAFR‐mediated responses by a translational modification of PAFR protein, and suggest that, in vivo, activation of Rho kinase by PAF may be a further pathway to sustain PAFR‐mediated PVSMC proliferation.

Introduction

Platelet‐activating factor, 1‐O‐alkyl‐2‐acetyl‐sn‐glyceryl‐3‐phosphorylcholine (PAF), is a potent phospholipid mediator that plays an integral role in a variety of biological processes 1 and it is an important mediator of the pulmonary circulation 2, 3, 4, 5, 6. In sheep in utero, endogenous PAF maintains high pulmonary vasomotor tone and vascular resistance in foetal lambs 4. Although rate of PAF catabolism by PAF acetylhydrolase is lower in foetal lamb lungs, pulmonary vascular PAF production is greater in the foetus than in the newborn, particularly in veins compared to arteries 7, 8. Combination of higher levels of PAF production in foetal pulmonary vasculature in vivo with lower rates of PAF catabolism results in availability of more PAF for binding to its receptors. The role of PAF in pathogenesis of hypoxia‐induced pulmonary vascular remodelling has been studied in a variety of animal models 9, 10, 11 and we have recently shown that in ovine foetal lung in utero, increased PAFR protein expression and PAFR binding contribute to pulmonary vascular remodelling, and may predispose them to persistent pulmonary hypertension after birth 9.

In the lung, PAF acts through its membrane‐bound receptors to evoke vasoconstriction 2, 4, 5, 6, 12, 13, 14 and it has been reported that PAF‐induced smooth muscle contraction is mediated by Rho‐kinase 15. RhoA is a member of the Rho family of low molecular weight G proteins, which regulate a variety of cell functions including cell growth, gene expression, Ca2+ sensitization and cytoskeletal rearrangement 16, 17 and RhoA activities are regulated by extracellular stimuli, which activate cell surface receptors 16, 18. Studies investigating involvement of RhoA in various cellular responses have been facilitated by generation of RhoA cDNA constructs and specific Rho kinase (ROCK) inhibitors 19, 20, 21. PAFR binding induces an intracellular signalling cascade, which leads to Ca2+ mobilization 7, 22; these reports suggest, in part, an interaction between PAF and RhoA pathways. Previous studies have shown that Rho kinase inhibitor, Y‐27632, reduces PAF‐induced pressor responses in isolated perfused lungs 15, but the mechanism by which Y‐27632 mediates PAF effects in vascular smooth muscles is not well understood. As Rho kinase is a down‐stream target of RhoA, we hypothesized that PAF‐induced proliferation of foetal ovine pulmonary vascular smooth muscle cells (PVSMC) would be mediated by RhoA/Rho kinase signalling. We carried out in vitro studies employing foetal ovine PVSMC to investigate involvement of RhoA/Rho kinase in their PAF‐receptor mediated proliferation and determined the role of RhoA/Rho kinase in PAF receptor expression and PAR‐mediated responses. We compared effects of Rho kinase inhibitors, Y‐27632 and Fasudil (HA‐1077) to PAF‐mediated responses, in smooth muscle cells from pulmonary arteries (SMC‐PA) and veins (SMC‐PV) to gain insight into regulatory pathways occurring in pulmonary arteries and veins of developing foetal lamb lungs.

Materials and methods

Materials

All studies were approved by the Institutional Animal Care and Use Committee of Los Angeles Biomedical Research Institute. Pregnant ewes (146–148 d gestation, term being 150 d) were purchased from Nebekar Farms (Santa Monica, CA, USA). Authentic standards of 1‐O‐hexadecyl‐2‐O‐acetyl‐sn‐glycero‐3‐phosphorylcholine [C16‐PAF (PAF)] and 1‐O‐hexadecyl‐sn‐glycero‐3‐phosphorylcholine (lyso‐C16‐PAF) as well as 1‐(5‐isoquinolinesulfonyl) homopiperazine, [Fasudil (HA‐1077)]; (R)‐(+)‐trans‐N‐(4‐pyridyl)‐4‐(1‐aminoethyl)‐cyclohexanecarboxamide, (Y‐27632) were purchased from Biomol (Plymouth Meeting, PA, USA). 3H‐thymidine was purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Phenylmethysulphonyl fluoride, leupeptin, pepstatin, bovine serum albumin, as well as actin antibody, were purchased from Sigma‐Aldrich Company (St. Louis, MO, USA) and antibody to PAFR was purchased from Cayman Chemical (Ann Arbor, MI, USA). All studies were conducted with freshly made reagents. Ecolite(+) liquid scintillation cocktail was purchased from MP Biochemicals (Irvine, CA, USA). All other reagents and chemicals were purchased from Fisher Scientific (Santa Clara, CA, USA).

Preparation of pulmonary vascular smooth muscle cells (PVSMC)

Intrapulmonary vessels were isolated from freshly killed term foetal lambs, then smooth muscle cells were harvested from the freshly excised arteries and veins under sterile conditions as previously reported 7. Cells were cultured and used at the 4 to 10th passages and identity of SMCs at each passage was characterized using smooth muscle cell‐specific monoclonal antibody (Sigma). SMC were devoid of endothelial cells and fibroblasts. Cell synthetic and proliferative phenotype did not change from 4 to 10th passages as has been shown in our previous reports 7, 23.

Experimental conditions

All studies were conducted in vitro on smooth muscle cells from intrapulmonary arteries and veins (SMC‐PA and SMC‐PV, respectively). Adherent cells were cultured in normoxia or under hypoxia, according to the specific experimental protocol. For normoxia, cells were studied in a humidified incubator at 37 °C aerated with 5% CO2 in air. Oxygen concentration was monitored with TED 60T per cent oxygen sensor (Teledyne Analytical Instruments, City of Industry, CA, USA). Incubator oxygen concentration was 21% and pO2 in culture media was maintained at 80–100 torr.

For hypoxia, an incubator set at 37 °C and first equilibrated for at least 1 h with gas mixture of 2% O2, 10% CO2 and balance N2, to maintain incubator culture media pO2 < 40 torr, determined on Nova Stat Profile 3 blood‐gas instrument, Nova Biomedical, Waltham, MA, USA 8. Cells were then placed in this incubator for experimentation and were continuously aerated with the hypoxic gas mixture throughout duration of the study. Oxygen concentration was monitored with TED 60T per cent oxygen sensor, as above.

Proliferation assay

Cells were seeded in 6‐well culture plates, 5 × 104 cells per well, and allowed to stabilize for 2–3 days. They were then starved by culturing in 0.1% FBS for 72 h and cultured in 10% FBS with or without test agents, in the presence of 5 μCi/well of 3H‐thymidine; they were then incubated for 24 h under normoxia or hypoxia according to the specific protocol. Proliferation assays were performed as previously reported 23. Test agents were dissolved in 10% FBS, which was also used as control for all culture conditions. After 24 h treatment, culture plates were placed on ice and culture medium was aspirated; cells were washed in ice‐cold PBS, followed by a wash in ice‐cold 5% trichloroacetic acid. At this point, cells labelled with 3H‐thymidine were extracted with 0.5 N NaOH and radioactivity of cell lysate was quantified on an LKB 6500 scintillation counter (Beckman Coulter, Fullerton, CA, USA). 3H‐thymidine was not added to cell proliferation studies quantified by cell counting.

Specific protocols

Effects of PAF receptor antagonists WEB 2170 on PAF‐induced cell proliferation were tested for in the presence and absence of PAF, as we have previously reported 23. Serum‐starved cells were pre‐incubated for 2 h in 10 μm WEB‐2170 dissolved in 10% the FBS growth medium or in 10% FBS growth medium alone. 10 nm PAF and 5 μCi of 3H‐thymidine were added and incubated for 24 h more under normoxia and hypoxia. 10% FBS controls received 5 μCi of 3H‐thymidine only, but were not treated with WEB 2170 nor 10 nm PAF test compounds.

Role of RhoA/Rho kinase (ROCK) inhibitors and mitogen‐activated protein kinase (MAPK) inhibitors on PAF‐induced cell proliferation

Effects of hypoxia, Y‐27632 and HA‐1077 on PAF stimulation of cell proliferation were studied. To determine that PAF stimulation of SMC‐PV, SMC‐PA proliferation occurred via the RhoA/Rho‐kinase pathway, serum‐deprived cells were pre‐incubated for 2 h under normoxia or hypoxia with 10 μm each of ROCK inhibitors Y‐27632 and Fasudil (HA‐1077); then 10 nm PAF and 5 μCi of 3H‐thymidine were added to each treatment sample and incubated further for 24 h under normoxia or hypoxia. The effect of 10% FBS culture medium alone was used as control for each study condition.

Effect of hypoxia, MAPK inhibitors PD 98059 and SB 203580 on PAF stimulation of cell proliferation

To determine the role of MAPK signalling in comparison with the RhoA/Rho kinase pathway in PAF stimulation of cell proliferation, we used MAPK inhibitors PD 98059 and SB 203580 to study PAF stimulation of SMC‐PV and SMC‐PA proliferation. Serum‐deprived cells were pre‐incubated for 2 h under normoxia or hypoxia with 30 μm each of PD 98059 and SB 203580; then 10 nm PAF and 5 μCi of 3H‐thymidine were added to each treatment and incubated further for 24 h under normoxia or hypoxia. The effect of 10% FBS culture medium alone was used as control for each study condition.

Transient cell transfection

Previous studies have demonstrated involvement of RhoA/ROCK in vascular responses of pulmonary arteries of rats 19, 24. Here, we found that the profile of effects of ROCK inhibitors was different between SMC‐PA and SMC‐PV with results on SMC‐PA being more distinct. Thus, we examined genetic modulation of PAFR‐mediated responses in SMC‐PA by investigating effects of RhoA cDNA constructs on PAF stimulation of SMC‐PA and SMC‐PV proliferation. Vectors encoding dominant negative RhoA(−/−) RhoA, mutated at residue 19, replacing threonine with asparagine and designated as T19N, and dominant positive (RhoA+/+) RhoA, mutated at residue 14, replacing glycine with valine, designated G14V. These plus pGFP control plasmid were purchased from the University of Missouri cDNA Resource Center (Rolla, MO, USA) and processed according the vendor's instructions on a Nucleofector™ II, with a nucleofector kit Amaxa biosystems (LONZA, Rockland, ME, USA). Briefly, cells were seeded in 6‐well culture plates at 50 000 cells/well and allowed to stabilize for 24 h. They were then treated with 1.5 μg/ml of each plasmid in lipofectamine transfection reagent and incubated for 48 h, after which transfection‐medium was replaced with fresh 10% FBS culture media. Transfection efficiency was between 20% and 25% within 24 h of transfection, as judged by pGFP fluorescence. Proliferative phenotype of transfected cells was compared to untransfected cells (as described above) for the cell proliferation assay. Effects of T19N, G14V vectors and pGFP control plasmids on cell proliferation were presented as 3H‐thymidine disintegrations per minute.

Effect of Y‐27632 on PAF receptor protein expression

Serum‐starved and sub‐confluent SMC‐PA and SMC‐PV were pulsed for 2 h under normoxia or hypoxia, with 10 μm Y‐27632, 10 μm HA‐1077, or 10% FBS growth medium. Then, 10 nm PAF was added to each set and incubated for 24 h more under normoxia or hypoxia. Cells incubated in 10% FBS alone for 24 h were used as control. After 24‐h incubation, proteins were prepared and PAFR protein expression was measured by western blotting and quantified against expression of GAPDH protein.

Western blotting

Western blotting was performed according to previous reports 23. Briefly, after incubation under hypoxia or normoxia, cells were washed in PBS and lysed in modified 40 mm HEPES hypotonic lysis buffer, pH 7.4, containing the following: 1 mm EGTA, 4 mm EDTA, 2 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, 0.1 mm phenylmethysulphonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μm 4‐(2‐aminoethyl) benzene sulphonyl fluoride, 200 mm sodium fluoride, 20 mm sodium pyrophosphate, 0.2 mm sodium vanadate and 0.1 mg/ml trypsin inhibitor. Proteins were recovered from lysed cells by centrifugation at 14 000g for 10 min in a refrigerated centrifuge and stored in 0.2 ml aliquots at −80 °C and used for western blotting.

SDS‐PAGE electrophoresis

Proteins were subjected to Coomassie blue quantification before use in western blotting with some modification of published methods 7, 25. Signals were developed using an Amersham ECL Western Blot detection kit then exposed to X‐ray film. Bands corresponding to PAFR protein or other proteins of interest were scanned using an UnscanIT program, to quantify blot density.

Rho kinase/PAFR expression immunocytochemistry

Pulmonary artery smooth muscle cells were grown on sterile microscope slides to 70–80% confluence then serum starved for 24 h with 0.05% FBS. On a study day, cells were washed in Ca2+/Mg2+‐free PBS then treated with 10 nm PAF or 10 μm Y‐27632 alone and 10 μm Y‐27632 + 10 nm PAF and incubated under normoxia or hypoxia for 24 h. After incubation, cells were washed and fixed for microscope study of PAF receptor expression, as reported previously 26. Briefly, cells were treated with 4% paraformaldehyde for 20 min, followed by washing in 0.1 m glycine and three more washings in PBS before being quenched by placing slides in 10% FBS in PBS for 30 min. Fixed cells were then incubated for 2 h at room temperature in 1:500 diluted monoclonal anti‐PAF receptor antibody (Cayman Ann Arbor, MI, USA), diluted in 10% FBS in PBS, then washed three times in 1% FBS in PBS. Next, cells were incubated for 30 min in 1:1000 dilution FITC‐conjugated goat anti‐rabbit secondary antibody (MP Biochemical, Irvine, CA, USA), also diluted in 10% FBS in PBS, then washed three times in 1% FBS in PBS. Slides were mounted using VectaShield, without propidium iodide staining. PAF receptor expression was localized using fluorescence microscopy with fluorescence images captured using a Zeiss Axioskop 40 instrument (Zeiss, Thornwood, NY, USA). For each slide, cells in four to five microscope views were counted to establish quantitative differences in PAFR protein expression in each condition. Cell counts from each study were averaged and subjected to sample statistics.

Data analysis

All numerical data are mean ± SEM. In all instances where radioisotope was used, background radioactivity was subtracted before quantifying radioactivity. Data were analysed using a two‐tailed t‐test followed by ANOVA (GraphPad Prism, San Diego, CA, USA). Results were considered significant at P < 0.05.

Results

PAF effect on cell proliferation

First we compared effects of 10% FBS and PAF on proliferation of arterial and venous smooth muscle cells respectively by cell counting, for normoxia and hypoxia conditions, and present these results in Fig. 1. In SMC‐PA, with 10% FBS, hypoxia caused 33% increase in cell proliferation. PAF treatment resulted in 75% increase under normoxia and 71% increase under hypoxia compared to 10% FBS, in each condition. In SMC‐PV, with 10% FBS, hypoxia caused 31% increase in cell proliferation. PAF treatment produced 45% increase compared to 10% FBS alone under normoxia; 21% increase compared to PAF treatment under normoxia, but 40% increase under hypoxia compared to 10% FBS under hypoxia. In general, PAF increased cell proliferation of each cell type and hypoxia further increased cell proliferation. Compared to SMC‐PA under the same conditions, proliferation by SMC‐PV was greater.

Figure 1.

Figure 1

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Effect of hypoxia and platelet‐activating factor ( PAF ) on pulmonary vascular smooth muscle cell proliferation. Data are mean ± SEM, n = 6. Serum‐starved cells were incubated under normoxia and hypoxia for 24 h with 10 nm PAF in 10% FBS or 10% FBS alone as control. Cell proliferation was quantified by direct cell counting. PAF increased proliferation of both SMC‐PA and SMC‐PV and hypoxia further increased proliferation. Statistical significance = *P < 0.05, difference from normoxia.

WEB 2170 inhibited PAF stimulation of PVSMC proliferation

Figure 2 shows the effect of PAF receptor antagonist, WEB 2170 (WEB), on proliferation of PVSMC (SMC‐PA and SMC‐PV) under normoxia and hypoxia. In SMC‐PA (panel A), 10% FBS culture medium alone, increased cell proliferation by 45% under hypoxia. Treatment of cells with PAF under normoxia significantly increased cell proliferation compared to 10% FBS alone, during normoxia. Hypoxia further increased cell proliferation compared to 10% FBS in normoxia or hypoxia, or the effect of PAF alone in normoxia. When cells were treated with WEB alone in normoxia, there was no difference in the effect of 10% FBS under normoxia. However, under hypoxia, WEB treatment produced a significant increase in cell proliferation compared to WEB alone in normoxia, however, this was less than for 10% FBS under hypoxia. Co‐incubation of WEB + PAF under normoxia produced no significant increase in cell proliferation compared to 10% FBS under normoxia and WEB alone under normoxia. Under hypoxia, co‐incubation of WEB + PAF produced a significant increase in cell proliferation compared to WEB + PAF normoxia and WEB alone under normoxia. However, during this effect of combined WEB and PAF treatment was still less than that of 10% FBS alone under hypoxia or that of PAF alone under normoxia or hypoxia. Thus WEB 2170 attenuated the effect of PAF at stimulating cell proliferation.

Figure 2.

Figure 2

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Effect of WEB 2170 on platelet‐activating factor ( PAF ) stimulation of pulmonary vascular smooth muscle cell proliferation under normoxia and hypoxia. Data are mean ± SEM, n = 6. Cells were pre‐incubated for 2 h in 10 μm WEB 2170 or in 10% FBS alone. Then, 10 nm PAF was added and incubation continued for 24 h more. Cells cultured for 24 h in 10% FBS alone were used as controls, while cells cultured in 10 nm PAF alone in 10% FBS were used as controls for PAF effect on proliferation. Proliferation was quantified as 3H‐thymidine uptake in disintegrations per minute (DPM). In general, WEB 2170 inhibited cell proliferation and combined WEB + PAF did not completely abrogate the effect of WEB 2170 on PAF stimulation of cell proliferation, in both cell types. Statistical significance = *P < 0.05, difference from normoxia; #P < 0.05, difference from effect of 10% FBS or PAF alone in each cell type.

In SMC‐PV (panel B), with 10% FBS, hypoxia caused increase in cell proliferation by 40% compared to under normoxic conditions. PAF treatment under normoxia increased cell proliferation by 45% over 10% FBS alone under normoxia. Hypoxia increased effect of PAF on cell proliferation compared to 10% FBS under normoxia or hypoxia and to PAF alone under normoxia. PAF stimulation of cell proliferation under hypoxia was 55% greater than under normoxia. In general, PAF treatment significantly increased cell proliferation over 10% FBS under normoxia and hypoxia. Treatment of cells with WEB alone under normoxia produced no significant change in cell proliferation compared to 10% FBS. Under hypoxia, the WEB effect on cell proliferation was not different from 10% FBS under normoxia, but was significantly less than of 10% FBS under hypoxia. Co‐incubation of WEB + PAF under normoxia did not reverse the inhibitory effect of WEB. Under hypoxia, combination of WEB + PAF increased cell proliferation by 50% compared to WEB alone and 10% FBS under normoxia, but this was still less than that of 10% FBS under hypoxia, as well as of PAF alone under normoxia and hypoxia. Thus, for both SMC‐PA and SMC‐PV, hypoxia augmented PAF stimulation of cell proliferation – which is in agreement with the results of cell counting – and proliferation was inhibited by PAF receptor antagonist WEB 2170.

Rho kinase inhibitors attenuated PAF stimulation of PVSMC proliferation

Figure 3 shows effects of Rho kinase inhibitors HA‐1077 and Y‐27632 on PAF stimulation of cell proliferation. In general, PAF treatment of both SMC‐PA and SMC‐PV under normoxia and hypoxia increased cell proliferation over that of 10% FBS alone. Figure 3a (panel A) indicates that treatment of SMC‐PA with HA‐1077 alone inhibited cell proliferation by 75% under normoxia and hypoxia compared to 10% FBS. Co‐incubation with PAF under normoxia produced no change in proliferation compared to HA‐1077 alone. However, HA‐1077 + PAF under hypoxia produced a non‐statistically significant difference in cell proliferation compared to 10% FBS alone under normoxia and hypoxia, but at the same time, proliferation was still less than that with PAF alone under normoxia or hypoxia. In SMC‐PV (panel B), HA‐1077 treatment under normoxia produced an effect similar to 10% FBS. Co‐incubation with PAF in normoxia did not increase cell proliferation compared to 10% FBS. Also, HA‐1077 treatment in hypoxia produced an effect similar to 10% FBS under hypoxia and co‐incubation of HA‐1077 and PAF in hypoxia significantly increased cell proliferation to the level PAF under normoxia, but less than PAF effect under hypoxia. Thus, HA‐1077 did not specifically inhibit PAF stimulation of cell proliferation.

Figure 3.

Figure 3

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Effect of Rho kinase inhibitors, HA‐1077 and Y‐27632 on platelet‐activating factor ( PAF ) stimulation of pulmonary vascular smooth muscle cell proliferation. Data are mean ± SEM, n = 5. Two sets of serum‐starved cells were pre‐incubated for 2 h in 10 μm each of Y‐27632 or HA‐1077, then 10 nm PAF was added to one set and all incubation continued for 24 h more under normoxia or hypoxia. (a: panels A and B) HA‐1077 alone inhibited proliferation under normoxia and hypoxia and inclusion of PAF did not produce any significant change in cell proliferation in SMC‐PA; however, SMC‐PV (panel B), effect was similar to 10% FBS alone. (b: panels A and B) Y‐27632 alone also inhibited cell proliferation under normoxia and hypoxia did not change the profile of cell proliferation in SMC‐PA; however, in SMC‐PV (panel B), the effect was similar to 10% FBS. Statistical significance = *P < 0.05, difference from the effect of PAF alone under normoxia; #P < 0.05, difference from the effect of 10% FBS under normoxia and hypoxia.

Figure 3b shows that treatment of SMC‐PA (panel A) with Y‐27632 alone inhibited cell proliferation by 80% under normoxia and hypoxia compared to 10% FBS. This is similar to the effect of HA‐1077 on SMC‐PA as seen in Fig. 3a (panel A). Co‐incubation of Y27632 and PAF in normoxia produced no change in proliferation compared to Y‐27632 alone. However, co‐incubation of Y‐27632 and PAF under hypoxia increased cell proliferation compared to that under normoxia, but the increase in proliferation was still less than that of 10% FBS under normoxia or hypoxia. With SMC‐PV (panel B), treatment with Y‐27632 alone increased cell proliferation by 30% compared to 10% FBS under normoxia. During hypoxia, Y‐27632 also significantly increased cell proliferation compared to 10% FBS in normoxia, but this was less than for 10% FBS in hypoxia. Co‐incubation of Y27632 + PAF in normoxia also increased cell proliferation compared to 10% FBS, but this was not different from that of Y‐27632 treatment alone under normoxia. However, under hypoxia, Y‐27632 + PAF treatment increased cell proliferation by 40% compared to Y‐27632 under hypoxia, and brought cell proliferation to the level of 10% FBS under hypoxia, with no significant difference to effect of PAF treatment alone under hypoxia. Thus, HA‐1077 and Y‐27632 each had different effects on cell proliferation of SMC‐PA and SMC‐PV under normoxia and hypoxia.

Figure 4 shows effect of dominant positive, RhoA(+/+) (G14N), and dominant negative, RhoA(−/−) (T19N), RhoA cDNA constructs, on PAF stimulation of SMC‐PA and SMC‐PV proliferation compared to the effect of 10% FBS and sham construct (pGFP control), under normoxia only. For SMC‐PA, Fig. 4a (panel A), treatment with G14V alone significantly increased cell proliferation, 60% compared to 10% FBS alone. Co‐culture of G14V and PAF also increased cell population growth, but there was no difference compared to G14V alone. Sham construct, pGFP control, also increased cell proliferation by 40%, but was not different from co‐culture pGFP and PAF.

Figure 4.

Figure 4

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Effect of RhoA (+/+) (G14V: a) and RhoA (−/−) (T19N: b) on platelet‐activating factor ( PAF )‐stimulation of proliferation studied in normoxia alone. Data are mean ± SEM, n = 5. Cell transfection was performed on serum‐starved cells as described in the Materials and methods section, then cell proliferation was compared to the effect of 10% FBS. G14V stimulated proliferation of SMC‐PA and SMC‐PV and co‐incubation with PAF produced no change in cell proliferation. T19N stimulated SMCPA proliferation (b: panel A), but had no effect on SMC‐PV and co‐incubation with PAF produced no change in cell proliferation compared to the agent alone. Statistical significance = *P < 0.05, difference from 10% FBS.

For SMC‐PV (panel B), treatment of cells with G14V construct alone also stimulated SMC‐PV proliferation by 30%. Co‐culture of G14V and PAF produced no change in cell proliferation compared to G14V alone. Unlike SMC‐PA, sham construct, pGFP control produced no significant change in cell proliferation compared to 10% FBS, but had less effect than G14V alone or G14V + PAF.

In Fig. 4b, T19N treatment alone increased SMC‐PA proliferation by 50% (panel A) compared to 10% FBS alone. However, the effect of T19N alone on cell proliferation was not different from that of T19N studied together with PAF. Treatment of cells with pGFP control plasmid alone or together with PAF also increased cell proliferation compared to 10% FBS, with no difference in effect of pGFP alone or pGFP + PAF.

For SMC‐PV (panel B), PAF increased their proliferation compared to 10% FBS alone. Treatment of cells with T19N alone or together with PAF did not produce any significant change in cell proliferation compared to 10% FBS. Also, pGFP treatment alone or together with PAF did not produce any statistically significant change in cell proliferation compared to 10% FBS alone or of T19N with or without PAF.

Figure 5 shows results of Rho kinase inhibitors on PAF receptor protein expression by PVSMC, designed to test effects of these inhibitors alone on their respective modulation of PAFR protein expression under normoxia and hypoxia. In SMC‐PA and SMC‐PV, Fig. 5a and 5b, respectively, upper panels show representative western blots of PAFR protein expression together with corresponding GAPDH protein standard for treatment conditions. Lanes 1–4 are normoxia (Nmx) identified as follows in protein/GAPDH ratio plots: lane1, control conditions under normoxia; lane 2, 10 nm PAF treatment; lane 3, 10 μm Y‐27632 alone; and lane 4, 10 μm HA‐1077 alone. Lanes 5–8 are repeats of lanes 1–4, but under hypoxic conditions. In SMC‐PA, Fig. 5a, treatment with 10 nm PAF for 24 h increased PAF receptor protein expression under normoxia (Nmx) and hypoxia (Hpx). Treatment with 10 μm each of Y‐27632 and HA‐1077 reduced PAF receptor protein expression during normoxia and hypoxia also. For SMC‐PV, Fig. 5b, PAF treatment also increased PAFR protein expression during normoxia (Nmx) and hypoxia (Hpx). However, as observed in SMC‐PA, both Y‐27632 and HA‐1077 treatments reduced PAFR protein expression during normoxia and hypoxia.

Figure 5.

Figure 5

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Effect of Rho kinase inhibitors on PAF receptor ( PAFR ) protein expression by pulmonary vascular smooth muscle cells. Data are mean ± SEM, n = 4. Serum‐starved cells were pre‐incubated for 2 h in 10% FBS, then were studied as described in the Materials and methods section Compared to controls in both cell types, PAF upregulated PAFR protein expression under normoxia (Nmx) and hypoxia (Hpx). Treatment with Y‐27632 and HA‐1077 attenuated PAFR protein expression. In both (a) SMC‐PA and (b), SMC‐PV statistical significance = *P < 0.05 difference from controls under normoxia (Nmx) or hypoxia (Hpx); #P < 0.05, difference from PAF treatment under normoxia and hypoxia; &P < 0.05, difference from control under normoxia and hypoxia.

Following our findings on effects of both HA‐1077 and Y‐27632, specially Y‐27632, on PAF stimulation of cell proliferation in SMC‐PA, we further investigated results of Y‐27632 on protein expression in SMC‐PA, by immunocytochemistry. Cells were quantified by counting their numbers per microscope field. Figure 6a shows qualitative photomicrographs of 10 μm Y‐27632 on PAF receptor expression by SMC‐PA by fluorescence microscopy; these were captured under the same gain and on the same plane, using a Zeiss Axioskop 40 microscope. Four to five microscope windows per slide were viewed to establish qualitative differences in PAFR protein expression in each condition. As shown in Fig. 6a, under normoxia, upper panel, PAF treatment increased PAFR protein fluorescence compared to 10% FBS control and Y‐27632 reduced PAFR fluorescence. Co‐incubation of cells with Y‐27632 + PAF not only reduced cell population per microscope field but also revealed disruption of cell cytoskeletons. Under hypoxia, lower panel, PAF treatment also increased cell population per view compared to 10% FBS control. Incubation with Y‐27632 alone reduced cell population compared to either 10% FBS control or PAF alone. Co‐incubation of Y‐27632 + PAF increased cell population compared to Y‐27632 alone, but when compared to PAF treatment alone, combined treatment not only reduced cell population per view, but also disrupted cell cytoskeletons, more so under hypoxia than in normoxia. Figure 6b shows quantification of immunocytochemical results. Hypoxia increased cell number in 10% FBS control compared to normoxia. Similar to observations in Fig. 1, PAF treatment increased cell number under normoxia and hypoxia compared to 10% FBS. Unlike effects of Y‐27632 treatment on cell proliferation, Y‐27632 treatment alone produced no change in PAFR fluorescence under normoxia and hypoxia compared to 10% FBS under normoxia, but inclusion of PAF increased PAFR fluorescence to 10% FBS under hypoxia.

Figure 6.

Figure 6

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Effect of Rho kinase inhibitor, Y‐27632, on PAF receptor ( PAFR ) protein expression studied in SMC ‐ PA under normoxia and hypoxia. Cells were pre‐incubated for 2 h under normoxia or hypoxia then cells were treated with reagents as shown in the legend. PAFR protein expression was probed as described in the Materials and methods section. (a) qualitative assessment, hypoxia and PAF increased PAFR protein fluorescence, but treatment with Y‐27632 not only reduced fluorescence but also disrupted cell membrane morphology. (b) PAF also increased cell number under both nomoxia and hypoxia, but Y‐27632 increased cell number to the level of 10% FBS in normoxia. Statistical significance = *P < 0.05, difference from normoxia; #P < 0.05, difference from 10% FBS under normoxia and hypoxia.

MAPK inhibitors inhibited PAF stimulation of PVSMC proliferation

Figure 7 shows effects of MAPK inhibitors PD 98059 and SB 203580 on PAF stimulation of cell proliferation. In general, PAF treatment in both SMC‐PA and SMC‐PV under normoxia and hypoxia increased cell proliferation over effects of 10% FBS alone. Figure 7a (panel A) reveals that treatment of SMC‐PA with PD 98059 alone produced no inhibition of cell proliferation under normoxia and hypoxia compared to 10% FBS. Co‐incubation of PD 98059 (PD) with PAF under normoxia or hypoxia significantly inhibited cell proliferation compared to PAF or 10% FBS alone under normoxia and hypoxia. In SMC‐PV (panel B), PD 98059 treatment was not different from 10% FBS in normoxia, but it reduced cell proliferation compared to 10% FBS under hypoxia. Co‐incubation of PD 98059 and PAF under normoxia and hypoxia inhibited PAF stimulation of cell proliferation compared to 10% FBS and PAF under normoxia and hypoxia. Thus, PD 98059 specifically inhibited PAF stimulation of cell proliferation in SMC‐PA and SMC‐PV.

Figure 7.

Figure 7

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Effect of inhibitors of MAPK signalling PD 98059 ( PD ) and SB 203580 ( SB ) on platelet‐activating factor ( PAF ) stimulation of cell proliferation. Data are mean ± SEM, n = 5. Two sets of serum‐starved cells were pre‐incubated for 2 h in 30 μm each of the inhibitor. 10 nm PAF was then added to one set of inhibitor‐treated cells and all incubation continued for 24 h under normoxia or hypoxia. In (a) and (b) (panels A and B), both PD and SB alone in SMC‐PA and SMC‐PV produced no change in cell proliferation particularly in normoxia, but co‐incubation of each inhibitor with PAF significantly prevented ability of PAF to stimulate cell proliferation compared to either 10% FBS control or PAF alone. Statistical significance = *P < 0.05 difference from normoxia; #P < 0.05 difference from effect of 10% FBS and PAF alone under normoxia and hypoxia.

Figure 7b shows that treatment of SMC‐PA (panel A) with p38 MAPK inhibitor SB 203580 (SB) alone did not alter levels of cell proliferation compared to 10% FBS. Under hypoxia, SB 203580 increased cell proliferation over 10% FBS, but less than the effect of PAF under hypoxia. Co‐incubation of SB + PAF significantly inhibited PAF stimulation of cell proliferation under normoxia and hypoxia. For SMC‐PV (panel B), treatment with SB 203580 under normoxia produced no change in cell proliferation compared to 10% FBS in normoxia, but reduced cell proliferation compared to 10% FBS under hypoxia. In both cases, SB 203580 was significantly less than that of PAF on cell proliferation. As observed for SMC‐PA, co‐incubation of SB+PAF significantly inhibited PAF stimulation of cell proliferation.

Discussion

During physiological growth and development, proliferation of PVSMC plays an important role in normal expansion of the vascular system 27. Proliferation of vascular smooth muscle cells is initiated by endogenous and exogenous stimuli, which may involve autocrine or paracrine mechanisms 28. Induction of cell proliferation by an agent entails activation of specific signals controlling cell division and cell growth and PAF, an endogenous lipid mediator, activates cell growth by both autocrine and paracrine mechanisms 29. Here we employed pharmacological manipulations in an in vitro setting under normoxia and hypoxia to examine any possible involvement of Rho kinase in PAF‐induced pulmonary vascular smooth muscle cell proliferation. The major finding of the study has been that Rho kinase inhibitors, Y‐27632 and HA‐1077 attenuated PAF stimulation of PVSMC proliferation under both normoxia and hypoxia, implicating Rho kinase as an endogenous modulator of PAF‐induced cell proliferation, by non‐specifically inhibiting expression of PAFR. Our data show that: (i) smooth muscle cells from pulmonary veins (SMC‐PV) proliferated more than those from pulmonary arteries (SMC‐PA) under normoxia and hypoxia and that their stimulation with PAF augmented cell proliferation in both conditions, in agreement with our previous report 23; (b) PAF induced proliferation of PVSMC via a PAF receptor‐specific pathway; (c) Results of experiments with WEB 2170 demonstrated that the effect of PAF on these cells occurred via PAF receptor‐mediated mechanisms and that inhibition of PAF stimulation of cell proliferation and PAFR protein expression suggest that Rho kinase inhibitors Y‐27632 and HA‐1077 inhibited PAF stimulation of proliferation by modulating PAFR expression, but not by specific inhibition of PAFR‐mediated signalling. Results of the G14V and T19N study suggest that RhoA neither inhibited nor stimulated PAF‐mediated responses, suggesting that Rho kinase modulated PAFR expression by transcriptional mechanism and modulated the PAFR‐mediated effect by post‐translational modification PAFR protein.

RhoA Rho kinase and PAFR‐mediated responses

Whether by cell counting or by DNA synthesis, proliferation of SMC‐PV was greater than of SMC‐PA, and although the western blot analysis was not designed to compare PAFR expression between the two cell types, PAFR expression by SMC‐PV was greater than by SMC‐PA. This suggests that greater PAFR expression of SMC‐PV under hypoxia was one reason for greater PAFR‐mediated effects. As shown in the immunocytochemistry study, Rho kinase inhibition of SMC‐PA resulted in changes in cell morphology, showing significant disruption in cell membrane homogeneity. This suggests that PAFR‐mediated responses in the cells were reduced as Rho kinase inhibitors prevented congenial binding of PAF to its receptor to effect downstream signal transduction. It is likely that the Rho kinase inhibitors disorganized salient PAF‐PAFR interaction, perhaps by creating conformational disorganization of PAFR protein, rather than by preventing direct binding of PAF to its membrane receptors. Additionally, results of western blotting suggest that the Rho kinase inhibitors alone acted to inhibit PAFR protein expression, perhaps by inhibiting translational processes. Quantification of cells of the immunocytochemical study showed that PAF treatment was unable to completely reverse inhibitory effects of Rho kinase inhibitor, implicating Rho kinase the enzyme, as attenuator of PAF‐mediated effects. This notion is supported by the fact that dominant negative, T19N and dominant positive G14V constructs of RhoA behaved similarly in promoting cell proliferation without augmentation of cell growth by co‐treatment with PAF. For SMC‐PV, effects of these constructs on PAF‐mediated cell proliferation were equally similar, although different from their effects on SMC‐PA. Of note is that of these constructs, especially T19N on proliferation of SMC‐PV where the effect was not different from that of sham control pGFP. Thus, absence of augmentation or attenuation of proliferation in the presence of PAF suggests that RhoA is not a specific gene modulator of PAFR‐mediated responses in SMC‐PA or SMC‐PV, but that the enzyme Rho kinase was the responsible molecule for regulating PAFR‐mediated responses so that inhibition of the enzyme attenuated the effect.

Physiological and pathological effects of PAF are mediated by its specific G protein coupled receptor, Gq 30, 31. We have shown in previous reports that PAF induces expression of its receptors in vivo and in vitro, suggesting that it activates intracellular molecules that regulate gene expression and cell growth 9, 23. Although hypoxia‐inducible factor‐1α (HIF‐1α) regulates vascular smooth muscle cell proliferation 32, 33, it has been shown that some other mitogens such as platelet‐derived growth factor, and epidermal growth factor (EGF) stimulate smooth muscle cell proliferation during hypoxia independent HIF‐1α 34, but EGF and VEGF modulate PAF stimulation of PVSMC proliferation 23. In this report, we used PVSMC to show a common observation of PAF effect in SMC‐PA and SMC‐PV in tandem. Previously, we showed that nuclear factor‐kappa B p65 (NF‐kB p65) and cyclin dependent kinases are downstream effectors of PAF‐induced PVSMC proliferation, with NF‐kB p65 presenting an important link between cytosolic and nuclear responses following PAF stimulation 23. The foregoing discussion portends that PAF activation of its membrane receptor induces intracellular signalling pathways that result in PVSMC growth and that interruption of this intracellular signal by either an endogenous or exogenous molecule, should interfere with normal PAF‐mediated responses. Furthermore, MAPK and EGF have been implicated as upstream mediators of NF‐kB p65 responses during PAFR‐mediated activity 23, 35, 36, 37. We now show that in both SMC‐PA and SMC‐PV, PAF is unable to abrogate effects of specific Erk1/2 inhibitor (PD 98059) and p38 inhibitor (SB 203580) of MAPK pathways. This is in agreement with previous results which showed MAPK signalling as one of the important downstream activators of mitogen‐activated cell proliferation 23, 35, 38, 39, 40. Our present findings suggest that Rho kinase may be a further protein that acts in the PAFR‐mediated signalling pathway to transduce protein activation signals to, perhaps, effect NF‐kB p65 translocation into the nucleus. Small GTPase, RhoA, and its effector protein, Rho kinase, are important regulators of vascular reactivity and general pulmonary physiology and pathophysiology 15, 20, 23, 24, 41, 42, 43, 44, 45. It has been reported that Rho kinase is a potent vasoconstrictor in the foetal pulmonary vascular bed, and that its action is via inhibition of action of nitric oxide synthase [NOS] 24, 28, 43. One important deduction of the inhibitory effect of Rho kinase on the NOS pathway in foetal circulation is that NOS inhibition by Rho kinase is necessary to maintain foetal pulmonary circulation in a constricted state, which is preferred for low foetal lung blood flow and lung development. However, a recent report by Alvira and associates 19 has shown that inhibition of Rho kinase with Y‐27632 resulted in pulmonary vasodilation in the ovine foetus even in the presence of NO. Our findings in ovine foetal smooth muscle cells presented here, suggest that one of the reasons for observed vasodilation following Rho kinase inhibition with Y‐27632 may in part, reside in inhibition of PAFR‐mediated responses. We have shown that in the ovine foetus, PAF acts through its receptors in the lung to maintain high pulmonary vasomotor tone in the hypoxic lung environment in vivo 4, and inhibition of the PAFR‐mediated effect resulted in reduced pulmonary artery pressure with concomitant increase in pulmonary blood flow. We have also shown that in vitro, PAF acts through its receptors to increase ovine foetal PVSMC proliferation and calcium release 6, 9. Thus, high PAFR‐mediated activity is necessary to maintain high pulmonary vasomotor tone in ovine foetal lungs. This indicates that inhibition of PAFR‐mediated effects in ovine foetal smooth muscle in vivo, even in a non‐specific mechanism, presents a detrimental condition for their lung function and development. Our findings that Rho kinase inhibitors reduce PAFR protein expression and PAFR‐mediated responses, specially under hypoxia, suggest the existence of a PAF‐PAFR‐Rho kinase interaction in foetal pulmonary circulation. Disruption of this congenital interaction would result in adverse foetal pulmonary circulatory conditions.

Abnormal regulation of functions of pulmonary vascular smooth muscle and its endothelium (that is, inability to downregulate smooth muscle cell‐ and endothelium‐derived vasoconstrictors, and upregulation of endothelium‐derived vasodilators) in the perinatal period, is implicated in pathogenesis of persistent pulmonary hypertension of the newborn [PPHN] 19, 46, 47. Therefore, downregulation of PAFR‐mediated responses postnatally, by inhibition of Rho kinase activity, may be one mechanism to prevent pulmonary vascular abnormalities of PPHN. Persistent PPHN is a pathological condition with different aetiologies. Neonates with PPHN have high PAF levels 48, showing that persistence of high PAF levels postnatally leads to abnormal perinatal pulmonary adaptation. We speculate that in vivo, in the hypoxic environment of foetal lungs where PAF level is high, PAF acts via its receptor in conjunction with activity of Rho kinase, to induce proliferation of smooth muscle cells, an important role in maintenance and remodelling of the foetal pulmonary vascular system during development and physiological growth in utero. We also speculate that uncontrolled cell growth postnatally, via inability to downregulate PAF receptor‐mediated effects, may lead to hyperplasia of pulmonary vascular smooth muscle resulting in incidence of PPHN.

Acknowledgements

This work was supported in part by grants 513292 by the Los Angeles Biomedical Research Institute, Torrance, CA and HL‐077819 from the National Heart Lung and Blood Institute NIH, Bethesda, Maryland.

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