Placental growth factor mediates aldosterone-dependent vascular injury in mice

Abstract

In clinical trials, aldosterone antagonists reduce cardiovascular ischemia and mortality by unknown mechanisms. Aldosterone is a steroid hormone that signals through renal mineralocorticoid receptors (MRs) to regulate blood pressure. MRs are expressed and regulate gene transcription in human vascular cells, suggesting that aldosterone might have direct vascular effects. Using gene expression profiling, we identify the pro-proliferative VEGF family member placental growth factor (PGF) as an aldosterone-regulated vascular MR target gene in mice and humans. Aldosterone-activated vascular MR stimulated Pgf gene transcription and increased PGF protein expression and secretion in the mouse vasculature. In mouse vessels with endothelial damage and human vessels from patients with atherosclerosis, aldosterone enhanced expression of PGF and its receptor, FMS-like tyrosine kinase 1 (Flt1). In atherosclerotic human vessels, MR antagonists inhibited PGF expression. In vivo, aldosterone infusion augmented vascular remodeling in mouse carotids following wire injury, an effect that was lost in Pgf-/- mice. In summary, we have identified PGF as what we believe to be a novel downstream target of vascular MR that mediates aldosterone augmentation of vascular injury. These findings suggest a non-renal mechanism for the vascular protective effects of aldosterone antagonists in humans and support targeting the vascular aldosterone/MR/PGF/Flt1 pathway as a therapeutic strategy for ischemic cardiovascular disease.

Figure 1. PGF expression is regulated by aldosterone via vascular MR activation.

Mouse aortas (A–C) or carotid arteries (D) were treated ex vivo with vehicle (white bars), aldosterone (black bars, 100 nM unless otherwise indicated), or aldosterone + spironolactone (gray bars, 10 μM spironolactone) for the indicated times. (A–C) Pgf mRNA expression was quantified by QRT-PCR of vessel RNA (A), Pgf protein was quantified by ELISA in vessel lysate (B), and secretion was quantified by ELISA of vessel conditioned media (C). (D) Carotid Pgf mRNA (left) and secreted protein (right) were quantified. ^#P < 0.05, ^##P < 0.001 versus vehicle and aldosterone + spironolactone. *P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle.

Figure 2. Mechanism of aldosterone regulation of PGF expression.

(A) Aldosterone regulates PGF transcription. Mouse aortas were treated ex vivo with vehicle (white bars) or aldosterone (black bars) in the presence of vehicle, 5 μg/ml actinomycin D (ActD), or 40 μg/ml CXMD, and Pgf mRNA expression was quantified by QRT-PCR of vessel RNA. *P < 0.001 versus vehicle, ^#P < 0.05 versus aldosterone treated with no inhibitor. (B) The PGF gene contains a functional MRE. HEK293 cells were transfected with a luciferase reporter driven by the PGF enhancer MRE in the absence (empty CMX vector) or presence of MR expression. The cells were treated with the indicated concentrations of aldosterone and/or 10 μM spironolactone (gray bars). Bars represent fold activation of normalized luciferase activity relative to vehicle-treated, similarly transfected cells. *P < 0.05, **P < 0.01, ***P < 0.001 versus MR plus 0 nM aldosterone and versus spironolactone alone. ^#P < 0.01 versus 100 nM aldosterone.

Figure 3. Endothelial injury enhances aldosterone-stimulated expression of PGF and its receptor, Flt1.

Intact and endothelium-denuded mouse aortas were treated ex vivo with aldosterone (A) or vehicle (V) for 8 hours. (A) Mouse aortic Pgf message was quantified by QRT-PCR. (B) Mouse Pgf secretion was quantified by ELISA using vessel-conditioned media. (C) Mouse aortic Flt1 and Flk1 messages were quantified by QRT-PCR. *P < 0.05 versus vehicle-treated and versus intact aldosterone-treated aortas.

Figure 4. PGF regulation by aldosterone and MR in atherosclerotic human vessels.

Normal and atherosclerotic human vessels (from CABG patients) were treated ex vivo with vehicle control (V), aldosterone (8 hours), or spironolactone (S) (1 μM, 18 hours). (A) Basal human PGF message was quantified by QRT-PCR. (B) Aldosterone induction of human aortic PGF and FLT1 messages were quantified by QRT-PCR. (C) Spironolactone effects on human PGF message was quantified by QRT-PCR. (D) Spironolactone effects on human PGF protein secretion was quantified by human PGF ELISA of vessel-conditioned media. ^†P < 0.05 versus normal vessel. ^#P < 0.05, ^##P < 0.01 versus vehicle-treated and versus aldosterone-treated normal vessel mRNA. *P < 0.05, **P < 0.001 versus vehicle-treated CABG vessel.

Figure 5. Aldosterone-enhanced SMC proliferation after vascular injury is inhibited in PGF-deficient mice.

Medial SMC proliferation was quantified in BrdU-stained sections of uninjured (U) and injured (I) WT and PGF KO mouse carotid arteries treated with vehicle or aldosterone. Medial VSMC BrdU labeling data for all animals is indicated at top. Representative carotid artery sections are shown at bottom (scale bar: 1 mm). *P < 0.01, **P < 0.001 versus uninjured.

Figure 6. Lack of aldosterone-enhanced ECM deposition after vascular injury in PGF-deficient mice.

ECM deposition was quantified in trichrome-stained sections of uninjured and injured WT and PGF KO mouse carotid arteries treated with vehicle or aldosterone. Fold change in medial trichrome pixel area for all animals compared with WT vehicle-treated, uninjured vessels is indicated at top. Representative carotid artery sections are shown at bottom (scale bar: 1 mM). *P < 0.001 versus uninjured.

Figure 7. Aldosterone-enhanced medial vessel thickening after vascular injury is inhibited in PGF deficient mice.

Medial vessel area was quantified in Elastin-stained sections of uninjured and injured WT and PGF KO mouse carotid arteries treated with vehicle or aldosterone. Medial area data for all animals is indicated at top. Representative carotid artery sections are shown at bottom (scale bar: 1 mm). *P < 0.05, **P < 0.001 versus uninjured.