Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

doi:10.1016/j.bbrc.2005.12.211

Biochemical and Biophysical Research Communications

Volume 341, Issue 2, 10 March 2006, Pages 591-598

https://doi.org/10.1016/j.bbrc.2005.12.211 Get rights and content

Abstract

Lectin-like oxidized LDL receptor-1 (LOX-1) is an oxidized LDL receptor, and its role in restenosis after angioplasty remains unknown. We used a balloon-injury model of rabbit aorta, and reverse transcription-polymerase chain reaction revealed that LOX-1 mRNA expression was modest in the non-injured aorta, reached a peak level 2 days after injury, and remained elevated until 24 weeks after injury. Immunohistochemistry and in situ hybridization showed that LOX-1 was not detected in the media of non-injured aorta but expressed in both medial and neointimal smooth muscle cells (SMC) at 2 and 24 weeks after injury. Low concentrations of ox-LDL (10 μg/mL) stimulated the cultured SMC proliferation, which was inhibited by antisense oligonucleotides of LOX-1 mRNA. Double immunofluorescense staining showed the colocalization of LOX-1 and proliferating cell nuclear antigen in human restenotic lesion. These results suggest that LOX-1 mediates ox-LDL-induced SMC proliferation and plays a role in neointimal formation after vascular injury.

Section snippets

Methods

Rabbit aorta model. Male Japanese white rabbits weighing 3.0–3.5 kg were used and all surgical procedures on rabbits were carried out under general anesthesia with sodium pentobarbital (40 mg/kg), ketamine (2 mg/kg), and xylazine (8 mg/kg). The balloon-injury model of the rabbit aorta was created as reported previously [9]. At various time points (6, 12, and 24 h, 2, 5, and 7 days, and 2, 4, 8, 16, and 24 weeks after balloon injury), animals were anesthetized with sodium pentobarbital (40 mg/kg),

Vascular injury model of the rabbit aorta

We used a balloon-injury model of rabbit aorta in which LOX-1 mRNA and protein expression levels were analyzed at 6, 12, and 24 h, 2, 5, and 7 days, and 2, 4, 8, 16, and 24 weeks after the balloon injury. Neointima appeared 5 days after balloon injury and the neointimal area increased until 8 weeks (data not shown). We have previously shown that not only media, but also the neointima, are composed predominantly of SMC based on immunohistochemical staining for muscle actin [9].

Time course of LOX-1 mRNA expression

Based on RT-PCR

Discussion

In this study, we demonstrated that LOX-1 mRNA and protein are induced and persistently expressed in neointimal and medial SMC of the rabbit aorta for 24 weeks after balloon injury. LOX-1 protein is similarly expressed in restenotic lesions after balloon-angioplasty in humans. In cell culture experiments, the chemical inhibitor of LOX-1 reduced the SMC proliferation stimulated by relatively low concentrations of ox-LDL which was hard to induce apoptosis, although previous studies have shown

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CD36 is required by nonclassical monocytes to effectively patrol the endothelium during early atherogenesis to limit inflammation and atheroma formation [57]. Similarly, LOX-1 is expressed on the cell membranes of vascular smooth muscle cells (VSMCs) and is upregulated by proinflammatory cytokines, such as TNF-α, interleukin-1 (IL-1) and interferon-gamma (INF-y) [58]. Binding of ox-LDL to LOX-1 stimulates the migration and proliferation of VSMCs.
Cardiovascular disease (CVD) is a major contributor to annual deaths globally. Atherosclerosis is a prominent risk factor for CVD. Although significant developments have been recently made in the prevention and treatment, the molecular pathology of atherosclerosis remains unknown. Interestingly, the recent discovery of proprotein convertase subtilisin/kexin type 9 (PCSK9) introduced a new avenue to explore the molecular pathogenesis and novel management strategies for atherosclerosis. Initial research focussed on the PCSK9-mediated degradation of low density lipoprotein receptor (LDLR) and subsequent activation of pro-inflammatory pathways by oxidised low density lipoprotein (ox-LDL). Recently, PCSK9 and lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) were shown to positively amplify each other pro-inflammatory activity and gene expression in endothelial cells, macrophages and vascular smooth muscle cells. In this literature review, we provide insight into the reciprocal relationship between PCSK9 and LOX-1 in the pathogenesis of atheroma formation and plaque instability in atherosclerosis. Further understanding of the LOX-1-PCSK9 axis possesses tremendous translational potential to design novel management approaches for atherosclerosis.
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Vascular smooth muscle cells (SMCs) are important vascular components that are essential for the regulation of vascular functions during vascular atherosclerogenesis and vascular injury. Oxidized low-density lipoprotein (oxLDL) is known to induce SMC activation and foam cell transformation. This study characterized the role of hepatoma-derived growth factor (HDGF) in oxLDL-induced foam cell formation in cultured primary rat aortic SMCs. OxLDL exposure significantly increased HDGF expression and extracellular release. It also upregulated atherogenic regulators in SMCs, including TLR4, MyD88, LOX-1, and CD36. Exogenous HDGF stimulation not only increased the expression of cognate receptor nucleolin, but also the innate immunity regulators TLR4/MyD88 and lipid metabolism regulators, including LOX-1 and CD36. Oil red O staining showed that HDGF did not initiate, but enhanced oxLDL-driven foam cell formation in SMCs. Further signaling characterization demonstrated that oxLDL evoked activation of PI3K/Akt and p38 MAPK signaling pathways, both of which were involved in the upregulation of HDGF, LOX-1, and CD36 induced by oxLDL. Gene knockdown experiments using LOX-1 targeted siRNA demonstrated that LOX-1 expression was critical for oxLDL-induced HDGF upregulation, while HDGF gene depletion completely abolished oxLDL-triggered TLR4, LOX-1, and CD36 overexpression and foam cell formation in SMCs. These findings strongly suggest that oxLDL-induced HDGF upregulation participates in subsequent LOX-1 and CD36 expression in aortic SMCs and mechanistically contributes to the formation of SMC-derived foam cells. The oxLDL/LOX-1/HDGF axis may serve as a target for anti-atherogenesis therapy.
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Blocking LOX-1 reduced oxLDL-induced production of hyaluronan in smooth muscles, protecting these cells from migration and adherence of monocytes to extracellular matrix [108]. Furthermore, LOX-1 has an important role in neointimal formation after vascular injury; during the progression of vascular injury, LOX-1 expression gradually shifts from cells in the media to the intima [16,106,109]. Platelets adhere to a dysfunctional endothelium and promote smooth muscle proliferation and migration [2,3].
The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for binding and uptake of oxidized low-density lipoprotein (oxLDL) in endothelial cells. LOX-1 is also expressed in macrophages, smooth muscle cells and platelets. Following internalization of oxLDL, LOX-1 initiates a vicious cycle from activation of pro-inflammatory signaling pathways, thus promoting an increased reactive oxygen species formation and secretion of pro-inflammatory cytokines. LOX-1 plays a pivotal role in the development of endothelial dysfunction, foam cell and advanced lesions formation as well as in myocardial ischemia. Furthermore, it is known that LOX-1 plays a pivotal role in mitochondrial DNA damage, vascular cell apoptosis, and autophagy. A large number of studies provide evidence of a LOX-1′s role in endothelial dysfunction, hypertension, diabetes, and obesity. In addition, novel insights into LOX-1 ligands and the activated signaling pathways have been gained. Recent studies have shown an interaction of LOX-1 with microRNA's, thus providing novel tools to regulate LOX-1 function. Because LOX-1 is increased in atherosclerotic plaques and contributes to endothelial dysfunction, several compounds were tested in vivo and in vitro to modulate the LOX-1 expression in therapeutic approaches.
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All these factors contribute to the recruitment and adhesion of monocytes to the activated endothelium, their penetration in the intima, their differentiation into macrophages and the formation of foam cells [103]. LOX-1 plays a crucial role in the biphasic signaling evoked by oxLDLs, particularly in the dual effect of oxLDLs on NF-κB [104], angiogenesis [105,106], SMC proliferation [107] or apoptosis [108]. LOX-1 stimulates adaptive, protective and antioxidant responses, mediated by the nuclear factor E2-related factor 2 (Nrf2) and the antioxidant stress protein heme oxygenase 1 (HO-1) in SMCs [109] and in endothelial cells [110].
The oxidative theory of atherosclerosis relies on the modification of low density lipoproteins (LDLs) in the vascular wall by reactive oxygen species. Modified LDLs, such as oxidized LDLs, are thought to participate in the formation of early atherosclerotic lesions (accumulation of foam cells and fatty streaks), whereas their role in advanced lesions and atherothrombotic events is more debated, because antioxidant supplementation failed to prevent coronary disease events and mortality in intervention randomized trials.
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Expression of lectin-like oxidized LDL receptor-1 in smooth muscle cells after vascular injury

Abstract

Section snippets

Methods

Vascular injury model of the rabbit aorta

Time course of LOX-1 mRNA expression

Discussion

Lancet

J. Mol. Cell. Cardiol.

Biochem. Biophys. Res. Commun.

FEBS Lett.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Mol. Cell. Cardiol.

Atherosclerosis

Atherosclerosis—an inflammatory disease

N. Engl. J. Med.

An endothelial receptor for oxidized low-density lipoprotein

Nature

Expression of lectin-like oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesions

Circulation

Roles of lectin-like oxidized LDL receptor-1 and its soluble forms in atherogenesis

Curr. Opin. Lipidol.

Overexpression of lectin-like oxidized low-density lipoprotein receptor-1 induces intramyocardial vasculopathy in apolipoprotein E-null mice

Circ. Res.

RAVEL Study Group, A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization

N. Engl. J. Med.

TAXUS-IV Investigators, A polymer-based, paclitaxel-eluting stent in patients with coronary artery disease

N. Engl. J. Med.