Cell selective cardiovascular biology of microsomal prostaglandin E synthase-1

Abstract

Background: Global deletion of microsomal prostaglandin E synthase 1 (mPGES-1) in mice attenuates the response to vascular injury without a predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell-specific differential use by prostaglandin synthases of the accumulated prostaglandin H(2) substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells, endothelial cells, and myeloid cells further to elucidate the cardiovascular function of this enzyme.

Methods and results: Vascular smooth muscle cell and endothelial cell mPGES-1 deletion did not alter blood pressure at baseline or in response to a high-salt diet. The propensity to evoked macrovascular and microvascular thrombogenesis was also unaltered. However, both vascular smooth muscle cell and endothelial cell mPGES-1-deficient mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery in comparison to their littermate controls. The hyperplasia was associated with increased proliferating cell nuclear antigen and tenascin-C expression. In contrast, the response to injury was markedly suppressed by myeloid cell depletion of mPGES-1 with decreased hyperplasia, leukocyte infiltration, and expression of proliferating cell nuclear antigen and tenascin-C. Conditioned medium derived from mPGES-1-deficient macrophages less potently induced vascular smooth muscle cell proliferation and migration than that from wild-type macrophages.

Conclusions: Deletion of mPGES-1 in the vasculature and myeloid cells differentially modulates the response to vascular injury, implicating macrophage mPGES-1 as a cardiovascular drug target.

Figure 1. Validation of mPGES-1 deletion in VSMCs and ECs

A and C, Real-time RT-PCR and western blot of mPGES-1 and COX-2 expression in cultured VSMCs (A) and ECs (C) from both SM22Cre/flox and Tie2Cre/flox mice. For RT-PCR, all samples were normalized to 18s rRNA (n=3; *p=0.05, nonparametric Mann-Whitney tests). For western blot, α-tubulin was used as a loading control. B and D, Prostanoid profile in VSMCs and ECs. PGE₂, 6-keto-PGF_1α (the stable hydrolysis product of PGI₂), PGD₂ and TxB₂ (the stable hydrolysis product of TxA₂) were detected by mass spectrometry in cultured VSMCs (B) and ECs (D) at baseline and after stimulation with IL-1β (n=3; *p=0.05, nonparametric Mann-Whitney tests).

Figure 1. Validation of mPGES-1 deletion in VSMCs and ECs

A and C, Real-time RT-PCR and western blot of mPGES-1 and COX-2 expression in cultured VSMCs (A) and ECs (C) from both SM22Cre/flox and Tie2Cre/flox mice. For RT-PCR, all samples were normalized to 18s rRNA (n=3; *p=0.05, nonparametric Mann-Whitney tests). For western blot, α-tubulin was used as a loading control. B and D, Prostanoid profile in VSMCs and ECs. PGE₂, 6-keto-PGF_1α (the stable hydrolysis product of PGI₂), PGD₂ and TxB₂ (the stable hydrolysis product of TxA₂) were detected by mass spectrometry in cultured VSMCs (B) and ECs (D) at baseline and after stimulation with IL-1β (n=3; *p=0.05, nonparametric Mann-Whitney tests).

Figure 2. Validation of mPGES-1 deletion in macrophages

Real-time RT-PCR (A) and western blot (B) of mPGES-1 and COX-2 expression in peritoneal macrophages from Mac-mPGES-1-WT (WT) or Mac-mPGES-1-KO (KO) mice. For RT-PCR, all samples were normalized to 18s rRNA (n=3; *p=0.05, nonparametric Mann-Whitney tests). C, Prostanoid profile in peritoneal macrophages. PGE₂, 6-keto-PGF_1α, PGD₂, and TxB₂ were determined by mass spectrometry in the culture medium (n=6; *p<0.05, unpaired t-tests with Welch’s correction; ***p<0.001, unpaired t-test).

Figure 3. Impact of mPGES-1 deletion in VSMCs or ECs on cardiovascular function

A, VSMC or EC deletion of mPGES-1 had no effect on blood pressure. 3 week high-salt diet elevated systolic blood pressure (SBP) similarly irrespective of genotype. (n=7-9; *p<0.05 vs. Baseline, paired t-tests) B, Heart rate was indistinguishable amongst the groups. C, Neither VSMC nor EC mPGES-1 deletion altered the time to thrombotic carotid artery occlusion after photochemical injury for both males (n=8-10) and females (n=5-10). D, Comparison of thrombogenesis in microscopic cremaster arterioles of SM22Cre/flox (n=8), Tie2Cre/flox (n=8) and mPGES-1-flox control mice (n=9). Median fluorescence intensity was plotted versus time after laser-induced injury of the cremaster arteriole vessel wall. No difference was observed in either VSMC or EC mPGES-1 deficient mice compared with controls.

Figure 4. Vascular mPGES-1 deletion accelerates intimal hyperplasia

A, Representative sections of hematoxylin eosin staining of sham-operated and wire-injured femoral arteries of SM22Cre/flox, Tie2Cre/flox and mPGES-1-flox mice 28 days after wire injury (bar=40μm). Intima to media ratio (B) and percentage stenosis (C) were significantly increased in both SM22Cre/flox and Tie2Cre/flox arteries (n=8-13; **p<0.01, unpaired t-tests with Welch’s correction for SM22Cre/flox vs. flox and without Welch’s correction for Tie2Cre/flox vs. flox; *p<0.05, ***p<0.001 vs. flox, unpaired t-tests). D, Representative immunohistochemical staining of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA) and tenascin-C (TN-C) in femoral arteries (bar=50μm).

Figure 4. Vascular mPGES-1 deletion accelerates intimal hyperplasia

A, Representative sections of hematoxylin eosin staining of sham-operated and wire-injured femoral arteries of SM22Cre/flox, Tie2Cre/flox and mPGES-1-flox mice 28 days after wire injury (bar=40μm). Intima to media ratio (B) and percentage stenosis (C) were significantly increased in both SM22Cre/flox and Tie2Cre/flox arteries (n=8-13; **p<0.01, unpaired t-tests with Welch’s correction for SM22Cre/flox vs. flox and without Welch’s correction for Tie2Cre/flox vs. flox; *p<0.05, ***p<0.001 vs. flox, unpaired t-tests). D, Representative immunohistochemical staining of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA) and tenascin-C (TN-C) in femoral arteries (bar=50μm).

Figure 5. Myeloid cell mPGES-1 deletion reduces intimal hyperplasia

A, Representative sections of hematoxylin eosin staining of sham-operated and wire-injured femoral arteries of Mac-mPGES-1-WT (WT) or Mac-mPGES-1-KO (KO) mice 28 days after wire injury (bar=40μm). Intima to media ratio (B) and stenosis (C) were significantly decreased in KO mice compared with WT controls (n=10; *p<0.05, **p<0.01, unpaired t-tests). D, Representative immunohistochemical staining of α-SMA, PCNA and TN-C in femoral arteries (bar=40μm). E, Representative immunohistochemical staining of CD45 in injured arteries from both vascular and macrophage mPGES-1 deletion mice (bar=40μm).

Figure 5. Myeloid cell mPGES-1 deletion reduces intimal hyperplasia

A, Representative sections of hematoxylin eosin staining of sham-operated and wire-injured femoral arteries of Mac-mPGES-1-WT (WT) or Mac-mPGES-1-KO (KO) mice 28 days after wire injury (bar=40μm). Intima to media ratio (B) and stenosis (C) were significantly decreased in KO mice compared with WT controls (n=10; *p<0.05, **p<0.01, unpaired t-tests). D, Representative immunohistochemical staining of α-SMA, PCNA and TN-C in femoral arteries (bar=40μm). E, Representative immunohistochemical staining of CD45 in injured arteries from both vascular and macrophage mPGES-1 deletion mice (bar=40μm).

Figure 6. VSMC proliferation and migration in response to conditioned media from cultured peritoneal macrophages

VSMCs from mPGES-1-flox mice were stimulated with Mac-mPGES-1-WT or Mac-mPGES-1-KO conditioned media (WT-CM or KO-CM, respectively) supplemented with or without 10μg/ml PDGF-BB or PGE₂ (50nM). A, PDGF-BB significantly increased cell proliferation in both WT-CM and KO-CM treated cells, while the proliferation was markedly suppressed in KO-CM treated cells (n=6; *p<0.05, **p<0.01, ***p<0.001, unpaired t-tests). B, Scratch-induced wound healing assay showed that the KO-CM treated cells migrated more slowly than WT-CM treated cells (n=5; *p<0.05, **p<0.01, unpaired t-tests) with PDGF-BB stimulation. C, Representative photos were taken at baseline (0h) and 18h after wounding, the starting and ending edge of cells were marked with dotted lines and solid lines, respectively. D, PGE₂ treatment completely rescued the impaired PDGF-BB stimulated proliferation in the KO-CM treated cells (n=6; *p<0.05, unpaired t-test). E, Scratch-induced wound healing. PGE₂ completely rescued the impaired PDGF-BB stimulated migration in the KO-CM treated cells (n=4; ***p<0.001, unpaired t-test). F, Representative photos were taken at baseline (0h) and 18h after wounding, the starting and ending edge of cells were marked with dotted lines and solid lines, respectively.

Figure 6. VSMC proliferation and migration in response to conditioned media from cultured peritoneal macrophages

VSMCs from mPGES-1-flox mice were stimulated with Mac-mPGES-1-WT or Mac-mPGES-1-KO conditioned media (WT-CM or KO-CM, respectively) supplemented with or without 10μg/ml PDGF-BB or PGE₂ (50nM). A, PDGF-BB significantly increased cell proliferation in both WT-CM and KO-CM treated cells, while the proliferation was markedly suppressed in KO-CM treated cells (n=6; *p<0.05, **p<0.01, ***p<0.001, unpaired t-tests). B, Scratch-induced wound healing assay showed that the KO-CM treated cells migrated more slowly than WT-CM treated cells (n=5; *p<0.05, **p<0.01, unpaired t-tests) with PDGF-BB stimulation. C, Representative photos were taken at baseline (0h) and 18h after wounding, the starting and ending edge of cells were marked with dotted lines and solid lines, respectively. D, PGE₂ treatment completely rescued the impaired PDGF-BB stimulated proliferation in the KO-CM treated cells (n=6; *p<0.05, unpaired t-test). E, Scratch-induced wound healing. PGE₂ completely rescued the impaired PDGF-BB stimulated migration in the KO-CM treated cells (n=4; ***p<0.001, unpaired t-test). F, Representative photos were taken at baseline (0h) and 18h after wounding, the starting and ending edge of cells were marked with dotted lines and solid lines, respectively.