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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.

SUBMITTER: Jaffe IZ

PROVIDER: S-EPMC2964966 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Placental growth factor mediates aldosterone-dependent vascular injury in mice.

Jaffe Iris Z IZ Newfell Brenna G BG Aronovitz Mark M Mohammad Najwa N NN McGraw Adam P AP Perreault Roger E RE Carmeliet Peter P Ehsan Afshin A Mendelsohn Michael E ME

The Journal of clinical investigation 20101101 11

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 aldos ...[more]

PMID: 20921624

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Project description:Study hypothesisPlacental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning.Study findingPGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development.What is known alreadyPGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf (-/-) mice.Study design, samples/materials, methodsHere, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay.Main results and the role of chancePgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf (-/-) brains. At E14.5, Pgf (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not.Limitations, reasons for cautionSince Pgf (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring.Wider implications of the findingsThese studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments.Large scale dataN/A.Study funding and competing interestsThis work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queen's University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.

Project description:Severe polycystic liver disease can complicate adult dominant polycystic kidney disease, a genetic disease caused by defects in polycystin-1 (Pkd1) or polycystin-2 (Pkd2). Liver cyst epithelial cells (LCECs) express vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2. We investigated the effects of VEGF on liver cyst growth and autocrine VEGF signaling in mice with Pkd1 and Pkd2 conditional knockouts.We studied mice in which Pkd1 or Pkd2 were conditionally inactivated following exposure to tamoxifen; these mice were called Pkd1(flox/-):pCxCreER (Pkd1KO) and Pkd2(flox/-):pCxCreER (Pkd2KO).Pkd1KO and Pkd2KO mice developed liver defects; their LCECs expressed VEGF, VEGFR-2, hypoxia-inducible factor (HIF)-1alpha, phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2), and proliferating cell nuclear antigen (PCNA). In Pkd2KO but not Pkd1KO mice, exposure to the VEGFR-2 inhibitor SU5416 significantly reduced liver cyst development, liver/body weight ratio, and expression of pERK and PCNA. VEGF secretion and phosphorylation of ERK1/2 and VEGFR-2 were significantly increased in cultured LCECs from Pkd2KO compared with Pkd1KO mice. Inhibition of protein kinase A (PKA) reduced VEGF secretion and pERK1/2 expression. Addition of VEGF to LCECs from Pkd2KO mice increased phosphorylated VEGFR-2 and phosphorylated mitogen signal-regulated kinase (MEK) expression and induced phosphorylation of ERK1/2; this was inhibited by SU5416. Expression of HIF-1alpha increased in parallel with secretion of VEGF following LCEC stimulation. VEGF-induced cell proliferation was inhibited by the MEK inhibitor U1026 and by ERK1/2 small interfering RNA.The PKA-ERK1/2-VEGF signaling pathway promotes growth of liver cysts in mice. In Pkd2-defective LCECs, PKA-dependent ERK1/2 signaling controls HIF-1alpha-dependent VEGF secretion and VEGFR-2 signaling. Autocrine and paracrine VEGF signaling promotes the growth of liver cysts in Pkd2KO mice. VEGF inhibitors might be used to treat patients with polycystic liver disease.

Project description:Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026.These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.

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Placental growth factor mediates aldosterone-dependent vascular injury in mice.

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Placental growth factor mediates aldosterone-dependent vascular injury in mice.

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