Neprilysin Regulates Pulmonary Artery Smooth Muscle Cell Phenotype Through a Platelet-Derived Growth Factor Receptor–Dependent MechanismNovelty and Significance
Reduced neprilysin (NEP), a cell surface metallopeptidase, which cleaves and inactivates proinflammatory and vasoactive peptides, predisposes the lung vasculature to exaggerated remodeling in response to hypoxia. We hypothesize that loss of NEP in pulmonary artery smooth muscle cells results in increased migration and proliferation. Pulmonary artery smooth muscle cells isolated from NEP−/− mice exhibited enhanced migration and proliferation in response to serum and platelet-derived growth factor, which was attenuated by NEP replacement. Inhibition of NEP by overexpression of a peptidase dead mutant or knockdown by small interfering RNA in NEP+/+ cells increased migration and proliferation. Loss of NEP led to an increase in Src kinase activity and phosphorylation of PTEN, resulting in activation of the platelet-derived growth factor receptor (PDGFR). Knockdown of Src kinase with small interfering RNA or inhibition with PP2, a src kinase inhibitor, decreased PDGFRY751 phosphorylation and attenuated migration and proliferation in NEP−/− smooth muscle cells. NEP substrates, endothelin 1 or fibroblast growth factor 2, increased activation of Src and PDGFR in NEP+/+ cells, which was decreased by an endothelin A receptor antagonist, neutralizing antibody to fibroblast growth factor 2 and Src inhibitor. Similar to the observations in pulmonary artery smooth muscle cells, levels of phosphorylated PDGFR, Src, and PTEN were elevated in NEP−/− lungs. Endothelin A receptor antagonist also attenuated the enhanced responses in NEP−/− pulmonary artery smooth muscle cells and lungs. Taken together our results suggest a novel mechanism for the regulation of PDGFR signaling by NEP substrates involving Src and PTEN. Strategies that increase lung NEP activity/expression or target key downstream effectors, like Src, PTEN, or PDGFR, may be of therapeutic benefit in pulmonary vascular disease.
Neprilysin (NEP) is a cell surface peptidase expressed in vascular cells, cardiac myocytes, lung, brain, renal epithelial cells, and fibroblasts.1,2 It catalyzes the degradation of various neuropeptides implicated in migration, proliferation, and contraction, including atrial natriuretic peptide, Enkephalins, substance P, bradykinin, endothelin 1, and angiotensin II.1 Among growth factors, fibroblast growth factor (FGF) is cleaved by NEP, but platelet-derived growth factor (PDGF) and transforming growth factor-β are not.3 In addition to its peptidase activity, basic amino acid residues in the tail of NEP contribute to protein–protein interactions, resulting in modulation of signaling factors in prostate cancer cell lines.4
NEP-deficient mice have an exaggerated response to inflammatory stimuli with colitis, septic shock, and pancreatitis-induced lung injury.5–7 We have shown that NEP null mice develop exaggerated pulmonary hypertension accompanied by increased muscularization of distal pulmonary arteries and medial and adventitial thickening in response to hypoxia.8
In the systemic circulation, inhibitors of NEP have a protective role and prevent fatty streak formation and atherosclerotic plaques in animal models of hypertension.9 It has been observed that an increase in NEP activity correlated with increased risk of hypertension, insulin resistance, and obesity in human subjects.10 Studies addressing mechanisms by which NEP regulates pulmonary vascular function could be important in determining the potential use of vasopeptidase inhibitors for systemic hypertension.
C57BL/6 NEP null mice were routinely used for isolation of pulmonary artery smooth muscle cells. In vivo studies were performed on NEP null mice on both C57BL/6 and FVB/n backgrounds.
Isolation and Characterization of PASMCs From Adult Mice
PASMCs were isolated from proximal medial tissue of the pulmonary artery from individual age-matched 13- to 17-week-old NEP+/+ and NEP−/− littermate mice, as described.8 In all of the experiments n represents the population of cells each isolated from different matched NEP+/+ and NEP−/−mice.
Boyden Chamber Assay
Migration was determined by a modified Boyden chamber assay using a polycarbonate filter with 8-µmol/L pore diameter obtained from Neuroprobe (Gaithersburg, MD).11 The cells that migrated to the lower surface were counted (5 random ×20 fields per well).
PASMCs were grown to confluence on 60-mm plates.12 The cells were serum starved in DMEM-F-12 medium with 0.2% serum for 24 hours. Quantitation of migration was done by counting the number of PASMCs in a 5-cm2 area of the scratch after 6 hours. The average from 3 different populations was used for statistical analysis.
3[H] Thymidine Incorporation
The effect of growth factors and neuropeptides on DNA synthesis was evaluated as reported previously.8
Cell lysates were prepared and proteins separated on SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with primary antibody and protein bands visualized by chemiluminescence.8 GAPDH was used as a loading control. A Bio-Rad gel scanner and densitometer (Gel DocXR with Quantity 1 program) were used to assess the intensity of the bands obtained by Western blots.
PASMCs were fixed with paraformaldehyde 2% and methanol and stained with antibodies to PDGF receptor (PDGFR) α and β. Cells were analyzed using a Gallios flow cytometer (Beckman Coulter) and Summit 4.3 software (Beckman Coulter). Ten-thousand events were obtained and analyzed for the percentage of gated singlets and mean intensity. Overlay plots were obtained using Kaluza software. Four different paired cell isolates were used for analysis.
PASMCs were transfected with lentivirus expressing human full-length NEP or peptidase dead mutant NEP (NEPX) at a multiplicity of infection of 10, as described previously.8
Small Interfering RNA Transfection
PASMCs were transfected with mouse specific small interfering RNA (siRNA; 10 nmol/L) or universal siRNA from Sigma Aldrich using Dharmafect Reagent from Dharmacon (Denver, CO) as per manufacturer recommendations. siRNA for NEP, PDGFRα, PDGFRβ, and Src were obtained from Sigma. Cells were used 48 hours after transfection for migration and proliferation assays.
Endothelin A Receptor Antagonist Treatment
PASMCs were treated with endothelin A receptor (ETAR) antagonist ambrisentan (10 µmol/L). NEP+/+ and NEP−/− mice on an FVB/n background were treated with the ETAR antagonist atrasentan (10 mg/kg) for 7 days in drinking water.
Data were analyzed using GraphPad Prism 4.02 for Windows (GraphPad Software for Science Inc, San Diego, CA). Results are presented as mean±SEM. The significance of differences between 2 measurements was determined by unpaired, 2-tailed t tests; 1-way ANOVA was used for multiple comparisons followed by Fisher least significance analyses. P<0.05 was considered statistically significant. The n for each experiment represents the number of cell isolates each obtained from a different mouse.
Loss of NEP Leads to Increased Migration and Proliferation of Mouse PASMCs
Migration and proliferation of PASMCs are important mechanisms contributing to pulmonary vascular remodeling.8,13 PASMCs from NEP−/− mice exhibited increased migration in the presence of serum (0.2%) and PDGF-BB (10 ng/mL) compared with wild-type cells assessed by wound healing and Boyden chamber assays (Figure 1A–1C). Proliferation was measured by 3H-thymidine incorporation at 3 different doses of serum and PDGF and showed 3- to 4-fold higher incorporation in NEP−/− PASMCs compared with NEP+/+ cells (Figure 1D and 1E).
Twelve of 15 different matched pairs of NEP+/+ and −/− PASMCs showed a major difference in proliferation on initial screening; these cell lines were used for further study. Enhanced smooth muscle cell outgrowth was also observed from NEP−/− pulmonary artery tissue cultured ex vivo compared with +/+ control (data not shown), suggesting that the differences observed in the phenotype were intrinsic to the pulmonary artery smooth muscle cell and not acquired over time in culture. Scratch assays performed in the presence of mitomycin (10 µmol/L) had no effect on the enhanced migration of NEP−/− PASMCs, suggesting that proliferation did not contribute (Figure S1A in the online-only Data Supplement).
Altering NEP Level Modifies Migration and Proliferation of PASMCs
NEP−/− PASMCs were infected with lentiviral vector expressing full-length human NEP. Migration and proliferation were measured after 48 hours. As seen in Figure 2A and 2B, replacing NEP in null cells attenuated migration and proliferation in the presence of serum (0.2%) and PDGF. The inset in Figure 2A shows levels of NEP expressed.
Inactivation of NEP in +/+ cells with a lentivirus expressing a peptidase dead mutant of human NEP (NEPX), with the inhibitor phosphoramidon, or knockdown of expression with siRNA to mouse NEP caused increased migration and proliferation in response to serum and PDGF similar to that observed with NEP−/− cells (Figure 2C–2F). There was a 4-fold increase in NEPX levels, as shown in Figure 2C. There was a 98±2% decrease in NEP expression after treatment with siRNA, as shown in Figure 2E. Inhibition of NEP with phosphoramidon enhanced migration and proliferation in NEP+/+ PASMCs similar to that observed with mutant NEPX (Figure S1B and S1C). NEPX had no effect on migration and proliferation in NEP−/− cells (data not shown), suggesting that the peptidase activity of NEP is required to inhibit the exaggerated responses in null cells and that substrates on NEP are mediating the effect.
Loss of NEP Increases PDGFR Expression and Signaling in PASMCs
To determine whether the enhanced migratory and proliferative responses to PDGF were due to an increase in receptor expression, we measured PDGFRα, β, and βY751 levels by flow cytometry (Figure 3A) and Western blot (Figure 3B and Figure S2A). Protein levels of PDGFRα, β, and activated βY751 were significantly higher in NEP−/− cells (Table S1). A decrease in mRNA for both receptors PDGFRα (0.67-fold) and β (0.56-fold) was observed in NEP null PASMCs, perhaps representing an autoregulatory mechanism to downregulate receptor expression (Figure S2B).
Src and PTEN influence PDGFR signaling by phosphorylation-dependent mechanisms and are also regulated by NEP.14,15 As shown in Figure 3B, levels of SrcY416 and PTENS380 were higher in NEP−/− PASMCs. Total PTEN levels trended lower (0.6-fold) in NEP−/− compared with +/+ cells but were variable and did not reach statistical significance.
siRNA-mediated knock down of NEP in wild-type PASMCs caused a 1.8-fold increase in phosphorylated (p) PDGFRY751, a 1.5-fold increase in p-Src, and a 1.2-fold increase in p-PTEN similar to that seen in null cells (Figure 3C). Conversely, lentiviral expression of NEP in −/− PASMCs attenuated PDGFR, Src, and PTEN phosphorylation (Figure 3D and 3E).
We compared the effect of PDGF treatment on phosphorylation of PDGFR, Src, and PTEN in NEP+/+ and NEP−/− PASMCs, as shown in Figure 3F and 3G. PDGFR phosphorylation in NEP+/+ cells was transient and increased at 15 and 30 minutes, whereas a more sustained rise is seen in NEP−/− PASMCs. Total levels of PDGFR were not affected by the treatment. Figure 3H shows that the level of p-Src was 2-fold higher and total PTEN lower at 24 hours in NEP−/− PASMCs.
Inhibition of PDGFR Attenuates Migration and Proliferation in PASMCs
PASMCs treated with siRNA to PDGFRα, β, and αβ were used to assess the contribution of the 2 receptors to migration and proliferation. As shown in Figure 4A, partial knockdown of each of the receptors was observed at 48 hours. Average expression levels from 3 different isolates were 55±15% for PDGFRα, 45±15% for β, and 25±20% for αβ siRNA. We cannot rule out some degree of antibody cross-reactivity to the 2 receptors contributing to the observed magnitude of each knockout.
We found that selective PDGFRβ, but not α, activation stimulated migration and proliferation in both cell types (Figure 4B and 4C and Figure S3A and S3B). Results obtained from siRNA-mediated knockdown of PDGFRα, β, and αβ show that PDGFRβ was the predominant receptor contributing to migration and proliferation of PASMCs (Figure 4B and 4C). Cells treated with siRNA to PDGFRα maintained their migratory and proliferative response to PDGF-BB (Figure 4B and 4C).
We assessed levels of phospho- and total Src and PTEN in PASMCs treated with siRNA to PDGFRα, β, and αβ. As seen in Figure 4D, knockdown of both PDGFRα and β caused a significant decrease in p-Src, with total levels remaining unchanged. In the case of PTEN, knockdown of PDGFR increased both p-PTEN and total PTEN levels. Total PTEN levels were 1.8-fold higher in cells treated with PDGFR αβ siRNA (Figure 4E).
We found that knockdown of PDGFR αβ had significant effects on Src kinase activation in PASMCs, suggesting that both receptors contribute to its activation (Figure 4D and 4E). We think that the residual p-Src is attributed to PDGFR-independent and neuropeptide-dependent activation, because PDGFRβ or αβ siRNA completely inhibited basal p-Src phosphorylation in NEP+/+ cells (data not shown). It is interesting that PDGFRα-associated Src activity does not influence migratory and proliferative responses, whereas PDGFRβ does, suggesting that the 2 receptors couple to different downstream effectors.
Pharmacological inhibition of the PDGFR kinase (PDGFR inhibitor III) attenuated migration and proliferation of NEP−/− PASMCs (Figures 4A and 4B and Figures S3A and S3B). Inhibition of PDGFR kinase activity did not show significant effects on Src phosphorylation in NEP−/− PASMCs, suggesting that activity of PDGFR was not required for Src activation and may be mediated by the NEP substrates (Figures S3C and S3D). We observed reduced p-PTEN and total PTEN in cells treated with PDGFR kinase inhibitor III. Additional studies are required to understand the regulation of PTEN by PDGFR.
Inhibition of Src Kinase Attenuates Migration and Proliferation in Mouse PASMCs
To determine the role of activated Src, we used siRNA to knock down Src and PP2, a Src kinase inhibitor, and measured effects on migration and proliferation. Western analysis confirmed a 90±10% decrease of Src protein with no significant changes in Fyn and Lyn (data not shown). As seen in Figure 5A and 5B, treatment of PASMCs with Src siRNA inhibited migration and decreased proliferation to baseline levels in NEP−/− cells. Similarly, PASMCs treated with the inhibitor, PP2 (10 µmol/L), had decreased migration and proliferation (Figure S4A and S4B). Knockdown of Src with siRNA reduced PDGFRY751 phosphorylation to baseline levels and increased PTEN phosphorylation (Figure 5C and 5D). Src siRNA also increased total PTEN levels. In comparison, inhibition of Src kinase with PP2 treatment abolished PDGFRY751 phosphorylation but did not have significant effects on p-PTEN (Figure S4C and S4D).
NEP Substrates Enhance PDGFR-Induced Migration and Proliferation in NEP+/+ Cells
Endothelin (ET)-1 and fibroblast growth factor 2 (FGF2) are representative substrates of NEP and have been demonstrated to play a role in the pathogenesis of pulmonary hypertension.16 Levels and stability of ET-1 in cell culture supernatants and FGF2 in cell lysates were measured at baseline and were found to be higher in NEP−/− PASMCs (Figure S5).
NEP+/+ PASMCs were treated with PDGF in the presence or absence of ET-1 or FGF2. ET-1 enhanced PDGF-induced migration significantly, which was attenuated by ambrisentan, an ET receptor antagonist (Figure 6A and 6B). FGF2 had more of a potentiating effect on the proliferative response of PDGF, which was attenuated by neutralizing antibody to FGF2 (Figure 6C and 6D). Inhibition of Src kinase with PP2 also attenuated the enhanced migratory response to PDGF plus ET-1 and the proliferative response to PDGF plus FGF2 (Figure 6B and 6D).
Neutralizing antibody to FGF2 inhibited proliferation but not migration of NEP−/− cells. In contrast, neutralizing antibody to PDGF attenuated migration of NEP null cells. There was also a trend toward an inhibitory effect on growth (Figure S5A–S5D).
ET-1 and FGF2 Enhance PDGF Signaling in NEP+/+ PASMCs by a Src-Dependent Mechanism
To further analyze the synergy between representative NEP substrates and PDGF signaling, NEP+/+ PASMCs were treated with PDGF in the presence or absence of ET-1 for 1, 2, 4, and 8 hours, and levels of phospho- and total PDGFR, Src, and PTEN were measured. The magnitude of p-PDGFR, p-Src, and p-PTEN activation was 1.3- to 1.5-fold higher and sustained with PDGF in the presence of ET-1 (Figure 7A).
Treatment of NEP+/+ PASMCs with ET-1 increased phosphorylation of PDGFR y751 by 1.3-fold and Src by 1.5-fold and was inhibited by ambrisentan and PP2 (Figure 7B and 7C). A time course of activation of PDGFR and Src by ET-1 is shown in Figure S7A. Treatment of NEP+/+ cells with FGF2 for 24 hours increased PDGFR phosphorylation by 1.3-fold and Src by 1.5-fold (Figure 6B and Figure S7B). Inhibition of Src eliminated FGF2-induced PDGFR phosphorylation (Figure 7D and 7E).
ETAR Antagonist Attenuates PDGF Responses in NEP−/− PASMCs and Lungs
ETAR antagonist ambrisentan attenuated both migration and proliferation of PDGF-stimulated NEP−/− cells (Figure 8A and 8B). Addition of ET-1 (10 nmol/L) further increased phosphorylation of Src and decreased PTEN levels (Figure 8C). Ambrisentan decreased PDGFR and PTEN phosphorylation both at baseline and in response to PDGF (Figure 8D and 8E). Src activity was inhibited only in response to PDGF. Analysis of lung lysates from NEP−/− versus NEP+/+ mice showed that p-PDGFR, p-Src, and p-PTEN were higher in the null tissue, suggesting that the pathway is activated in vivo. Treatment with ETAR antagonist atrasentan attenuated the baseline phosphorylation of PDGFR and Src and increased levels of PTEN (Figure 8F and 8G).
In this study we show that loss of the endopeptidase activity of NEP in PASMCs leads to increased migration and proliferation to growth stimuli. A peptidase-inactive mutant of NEP or knockdown with siRNA showed similar effects in wild-type cells, suggesting a role for NEP substrates, which include vasoconstrictors and dilators belonging to the G protein–coupled receptor family of ligands.
Our results demonstrate a mechanism by which NEP contributes to pulmonary vascular remodeling involving neuropeptide-mediated transactivation of growth factor receptor and signaling intermediates.17 In NEP−/− cells, increased local concentrations of NEP substrates cause phosphorylation of Src kinase, inactivation of PTEN, and constitutive activation of PDGFR, which is partially reversed by the ETAR antagonist ambrisentan (Figure 8). Enhanced PDGFR signaling was observed in NEP−/− lungs and was attenuated by a ETAR antagonist.
Representative NEP substrates, ET-1 and FGF2, synergize with PDGF in wild-type PASMCs and increase phosphorylation of Src kinase and PDGFR. This phosphorylation is inhibited by ambrisentan and neutralizing antibody to FGF2 (Figure 7). This study provides new insights into how ET receptor antagonists may exert some of their attenuating effects on vascular cell migration and growth (Figure 7).
PDGF and PDGFR are overexpressed in rodent models of pulmonary hypertension and in pulmonary arteries of patients with idiopathic pulmonary hypertension.18,19 In experimental models it has been shown that PDGFR signaling is suppressed by BMPR2 (bone morphogenetic protein receptor 2), peroxisome proliferator-activated receptor-γ, and PTEN, and expression of these proteins is frequently lost in pulmonary hypertension.20–22 Our results suggest a novel mechanism by which a peptidase suppresses PDGF signaling and maintains smooth muscle cells in a quiescent state. Loss of NEP in PASMCs leads to activation of Src, resulting in phosphorylation and expression of PDGFR (Figures 3 and 4).
A potential role for Src kinase has been described in the BMPR2 kinase–deficient model of pulmonary hypertension.23 In NEP null PASMCs, increased levels of NEP substrates, ET-1 and FGF2, caused Src activation and enhanced PDGFR signaling.24 Inhibition or knockdown of Src decreases phosphorylation of PDGFRY751 and attenuates migration and proliferation (Figure 5C–5E).
PTEN plays an important role in vascular biology, and its loss in mice causes pulmonary hypertension.25 We have found that PTEN is inactivated by phosphorylation and expressed at lower levels in NEP−/− PASMCs compared with wild-type cells. Expression of NEP in null cells attenuated the increased phosphorylation of PTEN (Figure 3D). In addition, siRNA-mediated knockdown of PDGFR or Src increases PTEN levels, whereas the kinase inhibitors decrease phosphorylation, suggesting an indirect regulatory mechanism (Figures 4E, 4F, 5C, and 5D and Figures S4 and S5).
Vasopeptidase inhibitors that target NEP have been shown to be beneficial in systemic hypertension but were associated with increased risk of angioedema.26,27 Our results suggest that NEP inhibition in the pulmonary circulation could predispose the lung vasculature to injury. The role of NEP in different tissues may depend on local substrates available and resident cell composition and phenotype. These factors may vary in different vascular beds, emphasizing the need for understanding the mechanism of NEP action in the lung.
We demonstrate here for the first time that NEP activity is required to maintain a quiescent pulmonary artery smooth muscle cell phenotype. This is consistent with our recent observations that NEP null mice have enhanced hypoxia-induced pulmonary vascular remodeling, and expression of NEP is decreased in lungs of patients with chronic pulmonary obstructive disease and pulmonary vascular remodeling.8,28 We also show that ET-A antagonist effects may be mediated at least in part by inhibition of PDGFR responses. Strategies to increase NEP activity and expression or to directly inhibit Src and PDGFR in the lung may be of therapeutic benefit for the prevention and treatment of pulmonary hypertension.
NEP is a cell surface enzyme that cleaves and inactivates both vasodilator and vasoconstrictor neuropeptides that are important for vascular function. Loss of NEP in PASMCs activates an integrated signaling network involving neuropeptides and growth factors, with Src kinase playing a central role. This cascade leads to increased migration and growth of PASMCs, an important component of vascular remodeling in pulmonary hypertension. Although NEP inhibitors may have protective effects in the systemic circulation, our observations also suggest that these drugs could predispose the lung vasculature to injury. Future studies will determine whether vascular bed-specific functions of NEP are attributed to intrinsic differences in cell types or to differences in substrates of NEP.
We thank Marian Maslak for expert general administrative and technical assistance and Dr Hanjun Guan for lentiviral constructs.
Sources of Funding
The work was supported by funding from National Heart, Lung, and Blood Institute (grants HL078927, HL014985, and HL095439) and VA Merit Review.
Preliminary results were presented in part at American Thoracic Society International Conferences 2010 and 2011, and published in abstract form (Am J Respir Crit Care Med. 2010;181:A1171 and Am J Respir Crit Care Med. 2011;183:A344).
The online-only Data Supplement is available with this article at http://hyper.ahajournals.org/lookup/suppl/doi:10.1161/HYPERTENSIONAHA.111.199588/-/DC1.
- Received May 29, 2012.
- Revision received December 12, 2012.
- Accepted January 10, 2013.
- © 2013 American Heart Association, Inc.
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Novelty and Significance
What Is New?
Loss of neprilysin (NEP) leads to integrated signaling of NEP substrates, Src, PTEN, and platelet-derived growth factor receptor and enhanced migration and proliferation of pulmonary artery smooth muscle cells. This unexpected finding explains why reduced NEP predisposes to exaggerated pulmonary hypertension.
What Is Relevant?
NEP is required for the maintenance of neuropeptide levels, regulation of vascular tone, and for keeping pulmonary artery smooth muscle cells in a quiescent state. Loss of NEP leads to increased migration and proliferation of pulmonary artery smooth muscle cells, important contributors to vascular remodeling in pulmonary hypertension. This study suggests new therapeutic approaches for the treatment of pulmonary hypertension.
This study demonstrates how loss of NEP activity leads to increased migration and proliferation of pulmonary artery smooth muscle cells involving cross-talk between G protein–coupled receptors and growth factors and an increase in the Src kinase and platelet-derived growth factor receptor signaling. Substrates of NEP, such as endothelin 1 and fibroblast growth factor, are currently therapeutic targets for the treatment of pulmonary hypertension. Our results suggest that NEP inhibitors could predispose the lung vasculature to injury by increasing levels of these peptides. Future studies will determine whether vascular bed-specific functions of NEP are attributed to intrinsic differences in cell types or differences in substrates of NEP.