Cyclooxygenase-2-derived prostaglandin E₂ promotes injury-induced vascular neointimal hyperplasia through the E-prostanoid 3 receptor

Abstract

Rationale: Vascular smooth muscle cell (VSMC) migration and proliferation are the hallmarks of restenosis pathogenesis after angioplasty. Cyclooxygenase (COX)-derived prostaglandin (PG) E₂ is implicated in the vascular remodeling response to injury. However, its precise molecular role remains unknown.

Objective: This study investigates the impact of COX-2-derived PGE₂ on neointima formation after injury.

Methods and results: Vascular remodeling was induced by wire injury in femoral arteries of mice. Both neointima formation and the restenosis ratio were diminished in COX-2 knockout mice as compared with controls, whereas these parameters were enhanced in COX-1>COX-2 mice, in which COX-1 is governed by COX-2 regulatory elements. PG profile analysis revealed that the reduced PGE₂ by COX-2 deficiency, but not PGI2, could be rescued by COX-1 replacement, indicating COX-2-derived PGE₂ enhanced neointima formation. Through multiple approaches, the EP3 receptor was identified to mediate the VSMC migration response to various stimuli. Disruption of EP3 impaired VSMC polarity for directional migration by decreasing small GTPase activity and restricted vascular neointimal hyperplasia, whereas overexpression of EP3α and EP3β aggravated neointima formation. Inhibition or deletion of EP3α/β, a Gαi protein-coupled receptor, activated the cAMP/protein kinase A pathway and decreased activation of RhoA in VSMCs. PGE₂ could stimulate phosphatidylinositol 3-kinase/Akt/glycogen synthase kinase3β signaling in VSMCs through Gβγ subunits on EP3α/β activation. Ablation of EP3 suppressed phosphatidylinositol 3-kinase signaling and reduced GTPase activity in VSMCs and altered cell polarity and directional migration.

Conclusions: COX-2-derived PGE₂ facilitated the neointimal hyperplasia response to injury through EP3α/β-mediated cAMP/protein kinase A and phosphatidylinositol 3-kinase pathways, indicating EP3 inhibition may be a promising therapeutic strategy for percutaneous transluminal coronary angioplasty.

Keywords: EP3; neointima formation; polarity; prostaglandin E2; vascular smooth muscle cell migration.

Figure 1. COX-2 deletion reduced vascular neointima formation in response to injury in mice

A, COX-1 and COX-2 expression in cultured VSMCs in the absence or presence of LPS. Left panel, representative western blots. Right panel, quantification of protein expression normalized to the levels of β-actin. *P < 0.05 vs. control without LPS treatment, n = 4. The results were repeated 3 times. B, RT-PCR analysis of mRNA expression of COX-1, COX-2, and mPGES-1 in cultured VSMCs in the absence or presence of LPS. *P < 0.05 vs. control without LPS treatment, n = 3. The results were repeated 3 times. C, Expression of COX-1 and COX-2 in the femoral artery in reponse to injury. White triangles indicate COX-2 staining in the perivasculature, red triangles indicate COX-1 staining in neointima at day 28 after injury. Scale bar, 50μm. D, Representative hematoxylin and eosin staining of crossections of wire-injured arteries from wild type (WT), COX-2 KO and COX-1>COX-2 mice. Scale bar, 50μm. E, Quantification of intima to media (I/M) ratio (left) and restenosis index (right) of post-injured arteries from WT, COX-2 KO and COX-1>COX-2 mice. *P < 0.05 vs. WT, n = 10-15.

Figure 2. Urinary PG metabolite profile in WT, COX-2 KO and COX-1>COX-2 mice before and 3 days after wire-injury

24-hour urine samples were collected from COX-2 KO, COX-1>COX-2 and WT male mice, and the levels of urinary PGD₂ metabolite-PGD-M (A), PGE₂ metabolite-PGE-M (B), PGI₂ metabolite PGI-M (C), TxA₂ metabolite-Tx-M (D) were measured. Data are presented as mean ± SEM, *P < 0.05 vs. WT controls, n = 10-15.

Figure 3. Pharmacological inhibition of EP3 receptor attenuates VSMC migration in vitro

A, RT-PCR analysis of the expression of EP1, EP2, EP3 and EP4 receptors in cultured VSMCs (left) and arteries (right). B, The effect of PGE₂ receptor antagonists on PGE₂-stimulated VSMC migration in the wound healing assay. The EP1 antagonist, SC-51322; the EP2 antagonist, AH6809; the EP3 antagonist, L-798, 106; the EP4 antagonist, L-161, 982. Representative phase-contrast images were taken at 0, 6, and 12 h after scratching. The wound area was measured and quantitated as described in the Methods section. Data are normalized by the wound area at the 0 timepoint. ** P < 0.01 vs. control, n = 3. C, The effect of different concentrations of L-798, 106 on PGE₂-stimulated VSMC migration in the wound healing assay. *P < 0.05 as indicated, n =4. D, The effect of PGE2 receptor antagonists on migration of VSMCs in a transwell assay. The number of migrating cells was normalized to the control group. *P < 0.05 vs. control, n = 3. E, The effect of EP3 antagonist L-798,106 on the migration of VSMCs. *P < 0.05 vs. control, n = 3.

Figure 4. EP3α and EP3β are involved in the mediation of VSMC migration and vascular remodeling in response to injury

A, EP3 deficiency significantly retarded VSMC migration. *P < 0.05 vs. WT, n = 3. B, Transwell migration analysis of the impact of re-expression of EP3α, EP3β, or EP3γ on the migration of the VSMCs isolated from EP3 KO mice. *P < 0.05 vs WT transfected with empty vectors, n = 3. C, Dose-dependent effect of transfected EP3α or EP3β expression vectors on VSMC migration. *P < 0.05 vs. EP3 KO transfected with empty vector or as indicated, n = 3. D, Representative hematoxylin and eosin staining of cross sections of wire-injured arteries from EP3 KO and WT mice 28 days after injury. Scale bar, 50μm. Quantification of intima to media (I/M) ratio (middle) and restenosis index (right) of injured arteries from EP3 KO and WT mice. *P < 0.05 vs. WT, n = 8-10. E, Representative hematoxylin and eosin staining of cross sections of wire-injured arteries from mice transduced with the lentivirus expressing each of the EP3 variants. F, Quantification of intima-to-media (I/M) ratio (left) and restenosis index (right). *P < 0.05, **P < 0.01 vs. control (WT infected by a GFP lentivirus), n = 8-10. Scale bar, 50μm.

Figure 5. Pharmacological inhibition or genetic deletion of EP3 impairs the polarization of VSMCs for directional migration

A, Representative confocal micrographs of primary post-confluent VSMCs isolated from WT and EP3 KO mice immunostained for γ-tubulin to localize the MTOCs (arrow) at 6 h after wounding in the presence or absence of the EP3 antagonist, L-798,106. Nuclei were stained with DAPI. The dashed box outlines the region enlarged to the right. Scale bar, 50μm. B, Schematic of the method used for quantifying the position of the MTOCs of wound-edge cells. C, Quantitation of front-polarized VSMCs from WT and EP3 KO mice, with or without L-798,106 treatment 6 h after wounding. *P < 0.05 vs. control for L-798,106 treatment or WT for EP3 KO, n = 3. Scale bar, 50μm. The results were repeated 3 times. D, Representative confocal micrographs of α-tubulin immunostaining of VSMCs from WT and EP3 KO mice, with or without L-798,106 treatment 6 h after wounding. Scale bar, 50μm. E, Representative confocal micrographs of F-actin (Left) immunostaining of VSMCs from WT and EP3 KO mice, with or without L-798,106 treatment 6 h after wounding. The white boxes outline the region enlarged to the lower panel. The number of cellular leading protrusions was quantitated as the number of leading protrusions divided by the number of cells at the leading edge (right). *P < 0.05 vs. control for L-798,106 treatment or WT for EP3 KO, n = 3. Scale bar, 50μm. The results were repeated 3 times.

Figure 6. EP3 deletion reduces activation of RhoA, Rac1, and Cdc42 in VSMCs

A, Western blot analysis of active (GTP-RhoA) and total RhoA in cultured VSMCs from WT and EP3 KO mice transfected with EP3α, EP3β or control vector (left). Densitometric quantification of GTP-RhoA levels normalized to the levels of total RhoA (right). *P < 0.05 vs. control, n = 6. B, Western blot analysis of Cdc42 (GTP-Cdc42), active Rac1 (GTP-Rac1), and total Cdc42 and Rac1 in cultured VSMCs from WT and EP3 KO mice transfected with EP3α, EP3β or control vectors as indicated (left). Densitometric quantification of Rac1 and Cdc42 activity normalized to that of total Rac1 and Cdc42 (right). *P < 0.05 vs. control. The data were collected from 3 independent experiments.

Figure 7. EP3α/β are coupled via Gαi protein and to regulate RhoA activity and VSMC migration through the cAMP/PKA pathway

A, Western blot analysis of binding of EP3α/β and Gαi. B, Cellular cAMP levels were measured in VSMCs isolated from EP3 KO mice transfected with EP3α, EP3β, or control vector constructs treated with an EP3 agonist M&B 28767(10 μmol/L) and adenylate cyclase (AC) activator forskolin (10 μmol/L) as indicated. *P < 0.05 vs EP3KO transfected with control vectors, n = 3. The results were repeated 3 times. C, PKA activity was measured in VSMCs from EP3 KO or WT mice transfected with EP3α, EP3β, or control vector constructs treated with or without the PKA inhibitor H-89 (10 μmol/L). Data are normalized to the values of the control group. *P < 0.05, n = 3. The results were repeated 3 times. D, Western blot analysis of active RhoA in VSMCs from EP3 KO and WT mice transfected with EP3α, EP3β, or control vector constructs with or without, pretreatment with the PKA inhibitor H-89 (10 μmol/L). E, Effect of H-89 on reorientation of MTOC of EP3 KO and WT VSMCs. *P < 0.05, n = 3. The results were repeated 3 times. F, Effect of H-89 on the migration of VSMCs from EP3 KO and WT mice transfected with EP3α, EP3β, or control vectors constructs. The number of migrating cells was normalized to the control group. *P < 0.05 as indicated, n = 4.

Figure 8. EP3α/β-mediated PI3K-Akt-GSK3β signaling axis modulates migration and polarization of VSMCs

A, Phosphorylation of Akt and GSK3β in VSMCs from EP3KO and WT mice in the presence or absence of LPS. B, The PI3K inhibitor Wortmannin (100 nmol/L) attenuated the phosphorylation of Akt and GSK3β induced by the overexpression of EP3α or EP3β in VSMCs isolated from EP3 KO mice. C, Wortmannin, SB216763(20 μmol/L) and HIMO (10 μmol/L) treatment reduced the upregulation of Rac1 activity in VSMCs overexpressing EP3α or EP3β isolated from EP3 KO mice. D, Wortmannin and LY294002 (20 μmol/L) treatment suppressed the upregulation of the Cdc42 activity in VSMCs overexpressing EP3α or EP3β isolated from EP3 KO mice, while HIMO and SB216763 treatment did not have effect. E, Wortmannin, HIMO and SB216763 treatment restrained MTOC reorientation (left) and transwell migration (right) by the EP3α or EP3β overexpression in VSMCs isolated from EP3 KO mice. MTOC reorientation: *P < 0.05, n = 3. The results were repeated 3 times. In transwell assay, the data were collected from 3 independent experiments, and the number of migrating cells was normalized to the control group.*P < 0.05 as indicated. F, Schematic diagram of EP3α/β-mediated polarization and directional migration of VSMCs through both cAMP/PKA and PI3K pathways upon PGE₂ stimulation.