Over-expression of Neuron-derived Orphan Receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury

Abstract

We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

Lysyl oxidase (LOX) limits VSMC proliferation and neointimal thickening through its extracellular enzymatic activity

“…Permanent ligation of the left common carotid artery was performed as previously described 20,41 . Four-month-old mice were distributed into four groups: WT (n = 12), WT mice receiving BAPN (100 mg/kg/day; ip; n = 7) from the day before surgical intervention and throughout the experimental procedure, TgLOX (n = 10) and TgLOX treated with BAPN (n = 6).…”

“…After 3 weeks under standard chow diet the animals were anaesthetized with ketamine (75 mg/kg) and medetomidine (1 mg/kg) and sacrificed by thoracotomy. Carotid arteries were then excised, fixed in 4% paraformaldehyde/0.1 M PBS (pH 7.4) for 24 h, and embedded in paraffin for morphometric and immunohistochemical analysis 41 . Serial sections (5 µm thick) of paraffin-embedded arteries were cut using a microtome (Leica JUNG RM 2055).…”

“…The media area is the area between EEL and IEL, and the intima area was calculated by subtracting the lumen area from the IEL area. The % stenosis was calculated as: [intima area/(intima area + lumen area)] × 100 20,41 . Measures corresponding to the three sections for each carotid artery were averaged.…”

“…Aortic VSMC from mouse were obtained by the explant technique as previously described 41 . Briefly, endothelium-denuded medial tissue was cut into 1 to 2 mm cubes that were transferred to a 25 cm 2 culture flask containing 5 mL of pre-warmed DMEM supplemented with 10% fetal calf serum (FCS; Biological Industries) and antibiotics (100 U/mL penicillin and 0.1 mg/mL streptomycin).…”

The neuron-derived orphan receptor 1 (NOR1) is induced upon human alternative macrophage polarization and stimulates the expression of markers of the M2 phenotype

“…Whereas Nur77 has been reported to mediate the pro-inflammatory response in macrophages via the NFkB pathway [16] by activating the inducible kinase IKKi [17], other studies reported that, similar as Nurr1, Nur77 may also exert anti-inflammatory effects in macrophages [15]. NOR1 has been suggested to be pro-atherogenic since it promotes human and murine VSMC and EC proliferation [18,19] and induces the expression of VCAM-1 and ICAM-1 in EC thus enhancing monocyte adhesion [20]. Moreover, macrophage NOR1 seems to play a protective role in atherogenesis, since it inhibits the inflammatory response of macrophages as well as their ability to transform into foam-cells [15].…”