Meizi Jiang

LR11, an LDL Receptor Gene Family Member, Is a Novel Regulator of Smooth Muscle Cell Migration

Abstract— LR11, a member of the LDL receptor family, is highly expressed in vascular smooth muscle cells (SMCs) of the hyperplastic intima, and induces enhanced migration of SMCs in vitro via its upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. In this study, we have delineated the mechanism by which LR11 elevates the expression levels of uPAR in SMCs. Secretion of soluble LR11 is induced in SMCs during the rapidly proliferating phase, and the secreted LR11 induces the migration activities of SMCs. Both the cell-anchored and secreted forms of LR11 have the capacity to bind to and form complexes with uPAR. LR11-overexpressing cells show significantly enhanced uPAR binding, but decreased uPAR internalization. LR11 colocalizes with uPAR on the cell surface and inhibits the LDL receptor–related protein (LRP)-mediated binding and internalization of uPAR. Thus, LR11 mediates the uPAR localization to the plasma membrane. LR11 is highly expressed in the atheromatous plaque areas of apoE knockout mice, particularly in the intimal SMCs at the border between intima and media. The neutralization of LR11 function with anti-LR11 antibody reduced cuff-induced intimal thickness in mice. The novel mechanism of regulation of uPAR localization in SMCs accompanied with enhanced migration activity possibly constitutes an important factor in the process of atherosclerosis and arterial remodeling.

A Secreted Soluble Form of LR11, Specifically Expressed in Intimal Smooth Muscle Cells, Accelerates Formation of Lipid-Laden Macrophages

Objective—Macrophages play a key role in lipid-rich unstable plaque formation and interact with intimal smooth muscle cells (SMCs) in early and progressive stages of atherosclerosis. LR11 (also called sorLA), a member of low-density lipoprotein receptor family, is highly and specifically expressed in intimal SMCs, and causes urokinase-type plasminogen activator receptor-mediated degradation of extracellular matrices. Here we investigated whether the secreted soluble form of LR11 (solLR11) enhances adhesion, migration, and lipid accumulation in macrophages using animal models and cultured systems. Methods and Results—Immunohistochemistry showed solLR11 expression in thickened intima of balloon-denuded rat artery. Macrophage infiltration into the cuff-injured artery was markedly reduced in LR11-deficient mice. In vitro functional assays using THP-1-derived macrophages showed that solLR11 (1 &mgr;g/mL) significantly increased acetylated low-density lipoprotein uptake by THP-1 cells and cell surface levels of scavenger receptor SR-A 1.7- and 2.8-fold, respectively. SolLR11 dose-dependently increased the migration activity of THP-1 macrophages and adhesion to extracellular matrices 2.0- and 2.1-fold, respectively, at 1 &mgr;g/mL. These effects of solLR11 were almost completely inhibited by a neutralizing anti-urokinase-type plasminogen activator receptor antibody. Conclusion—SolLR11, secreted from intimal SMCs, regulates adhesion, migration, and lipid accumulation in macrophages through activation of urokinase-type plasminogen activator receptor. The formation of lipid-laden macrophages in atherosclerotic plaques possibly is regulated by SolLR11 of intimal SMCs.

Ang II–stimulated migration of vascular smooth muscle cells is dependent on LR11 in mice

Medial-to-intimal migration of SMCs is critical to atherosclerotic plaque formation and remodeling of injured arteries. Considerable amounts of the shed soluble form of the LDL receptor relative LR11 (sLR11) produced by intimal SMCs enhance SMC migration in vitro via upregulation of urokinase-type plasminogen activator receptor (uPAR) expression. Here, we show that circulating sLR11 is a novel marker of carotid intima-media thickness (IMT) and that targeted disruption of the LR11 gene greatly reduces intimal thickening of arteries through attenuation of Ang II-induced migration of SMCs. Serum concentrations of sLR11 were positively correlated with IMT in dyslipidemic subjects, and multivariable regression analysis suggested sLR11 levels as an index of IMT, independent of classical atherosclerosis risk factors. In Lr11-/- mice, femoral artery intimal thickness after cuff placement was decreased, and Ang II-stimulated migration and attachment of SMCs from these mice were largely abolished. In isolated murine SMCs, sLR11 caused membrane ruffle formation via activation of focal adhesion kinase/ERK/Rac1 accompanied by complex formation between uPAR and integrin alphavbeta3, a process accelerated by Ang II. Overproduction of sLR11 decreased the sensitivity of Ang II-induced activation pathways to inhibition by an Ang II type 1 receptor blocker in mice. Thus, we demonstrate a requirement for sLR11 in Ang II-induced SMC migration and propose what we believe is a novel role for sLR11 as a biomarker of carotid IMT.

Development of an Immunoassay for the Quantification of Soluble LR11, a Circulating Marker of Atherosclerosis

BACKGROUND Vascular smooth muscle cells (SMCs) migrate from the arterial media to the intima in the progression of atherosclerosis, and dysfunction of SMCs leads to enhanced atherogenesis. A soluble form of the LDL receptor relative with 11 ligand-binding repeats (sLR11) is produced by the intimal SMCs, and the circulating concentrations of sLR11 likely reflect the pathophysiological condition of intimal SMCs. Furthermore, polymorphism of the LR11 gene has been found to be related to the onset of Alzheimer disease. This study describes the development of a sandwich immunoassay for quantifying sLR11 in human serum and cerebrospinal fluid. METHODS We used synthetic peptides or DNA immunization to produce monoclonal antibodies (MAbs) A2-2-3, M3, and R14 against different epitopes of LR11. RESULTS sLR11 was immunologically identified as a 250-kDa protein in human serum and cerebrospinal fluid by SDS-PAGE separation, and was purified from serum by use of a receptor-associated protein and MAb M3. An immunoassay for quantification of sLR11 with a working range of 0.25-4.0 microg/L was developed using the combination of MAbs M3 and R14. Treatment of serum with 5.25% n-nonanoyl-N-methyl-d-glucamine reduced the matrix effects of serum on the absorbance detection in the ELISA system. The linear dynamic range of the ELISA spanned the variation of circulating sLR11 concentrations in individuals with atherosclerosis. CONCLUSIONS A sandwich ELISA was established for quantifying sLR11 in serum and cerebrospinal fluid. This technique provides a novel means for assessing the pathophysiology of atherosclerosis, and possibly neurodegenerative diseases.

Enhanced circulating soluble LR11 in patients with coronary organic stenosis.

LR11, an LDL receptor family member, is expressed in intimal smooth muscle cells. It was found that the soluble form of LR11 (sLR11) is detected in serum, and the circulating sLR11 levels are positively correlated with intima-media thickness of carotid arteries in dyslipidemic subjects. To clarify the significance of serum sLR11, the circulating sLR11 levels in patients with organic coronary stenosis and the contributing risk factors for them were studied. The subjects, 150 patients with symptoms of coronary artery disease, underwent coronary angiographic examination, and were divided into sex- and age-matched two groups; one is organic coronary stenosis group (OCS) and the other is normal coronary group (NC). Serum sLR11 levels were significantly higher in OCS than in NC (4.9+/-2.7 U vs 3.6+/-1.8 U, p<0.05). Multivariate regression analysis showed that circulating sLR11 is independent contributing factor for the OCS, as well as diabetes mellitus and dyslipidemia. Among various coronary risk factors for sLR11 level, HbA1c showed the highest correlation coefficient (p<0.01). These results suggest that the circulating sLR11 might reflect coronary organic stenosis, and that hyperglycemic condition might be promoting factor for expression of LR11 in intimal smooth muscle cells.

Soluble LR11/SorLA represses thermogenesis in adipose tissue and correlates with BMI in humans.

Thermogenesis in brown adipose tissue (BAT) is an important component of energy expenditure in mammals. Recent studies have confirmed its presence and metabolic role in humans. Defining the physiological regulation of BAT is therefore of great importance for developing strategies to treat metabolic diseases. Here we show that the soluble form of the low-density lipoprotein receptor relative, LR11/SorLA (sLR11), suppresses thermogenesis in adipose tissue in a cell-autonomous manner. Mice lacking LR11 are protected from diet-induced obesity associated with an increased browning of white adipose tissue and hypermetabolism. Treatment of adipocytes with sLR11 inhibits thermogenesis via the bone morphogenetic protein/TGFβ signalling pathway and reduces Smad phosphorylation. In addition, sLR11 levels in humans are shown to positively correlate with body mass index and adiposity. Given the need for tight regulation of a tissue with a high capacity for energy wastage, we propose that LR11 plays an energy conserving role that is exaggerated in states of obesity.

SORL1 genetic variants and cerebrospinal fluid biomarkers of Alzheimer’s disease

The neuronal sortilin-related receptor with A-type repeats (SORL1, also called LR11 or sorLA) is involved in amyloidogenesis, and the SORL1 gene is a major risk factor for Alzheimer’s disease (AD). We investigated AD-related CSF biomarkers for associations with SORL1 genetic variants in 105 German patients with mild cognitive impairment (MCI) and AD. The homozygous CC-allele of single nucleotide polymorphism (SNP) 4 was associated with increased Tau concentrations in AD, and the minor alleles of SNP8, SNP9, and SNP10 and the haplotype CGT of these SNPs were associated with increased SORL1 concentrations in MCI. SNP22 and SNP23, and the haplotypes TCT of SNP19-21-23, and TTC of SNP22-23-24 were correlated with decreased Aβ42 levels in AD. These results strengthen the functional role of SORL1 in AD.

Matrix metalloproteinase-3 enhances the free fatty acids-induced VEGF expression in adipocytes through toll-like receptor 2.

Infiltrated macrophages (Mϕ) are believed to cause pathological changes in the surrounding adipocytes through the secretion of active molecules in visceral fat. Matrix metalloproteinase (MMP)-3 is secreted from Mϕ, and enhances expression of the inflammatory cytokines through the activation of toll-like receptor (TLR) 2. Visceral adipocytes express high levels of vascular endothelial growth factor (VEGF), and the degree of visceral fat accumulation is associated with the plasma VEGF concentration in obese subjects. The aim of the study is to clarify the role of MMP-3 in the enhancement of the free fatty acids (FFAs)-induced VEGF expression through TLR2 in visceral adipocytes. One mM FFAs induced VEGF mRNA and protein expression in 3T3-L1 adipocytes. The FFAs-induced VEGF expression was mostly mediated by TLR2. A high fat intake increased the VEGF mRNA expression in visceral fat and the VEGF concentration in plasma, accompanied with the increase in the plasma FFAs concentration in mice. These increases were largely inhibited in TLR2-deficient mice. The FFAs-induced VEGF expression was increased in the presence of Mϕ-conditioned medium (CM) in adipocytes, and the enhancement was inhibited by a MMP-3 inhibitor or a neutralizing antibody against MMP-3. The active form of MMP-3 induced the VEGF mRNA expression, as well as TLR2, in adipocytes. The increase in the VEGF expression by MMP-3 was inhibited by the treatment with siRNA for TLR2. The enhancement of FFAs-induced TLR2 expression by Mϕ-CM was inhibited by blocking of the MMP-3. The increase in the VEGF mRNA expression by Mϕ-CM or MMP-3 was partially inhibited by a neutralizing antibody against TNF-α. These results indicate that MMP-3 in Mϕ-CM enhances the FFAs-induced VEGF expression in adipocytes through the increase in the TLR2 expression. The MMP-3 secreted from the infiltrated Mϕ may be a regulator of the VEGF expression in visceral adipocytes.

Macrophages regulate tumor necrosis factor-alpha expression in adipocytes through the secretion of matrix metalloproteinase-3.

Objective:Adipocytes accumulated in the visceral area change their function to induce tumor necrosis factor-α (TNF-α) secretion with concomitant matrix metalloproteinase (MMP)-3 induction in mice. This study was performed to clarify the role of macrophages (Mφ)-secreted MMP on the functional changes in adipocytes using a culture system.Design:Cultures of 3T3-L1 adipocytes with THP-1 Mφ or the Mφ-conditioned medium were used to investigate the role of Mφ-MMP on the TNF-α gene in 3T3-L1 adipocytes by the addition of MMP inhibitors. For animal experiments, male C57BL/6J mice were rendered insulin resistant by feeding a high-fat diet, and the expression of an Mφ marker F4/80, and MMP-3 genes in mesenteric and subcutaneous fat tissue specimens were examined.Results:Mφ-conditioned media (Mφ-CM) increased the levels of TNF-α mRNA expression in 3T3-L1 adipocytes, and these adipocyte responses were abolished by treatment with GM6001, a broad-spectrum MMP inhibitor, or NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), an MMP-3 inhibitor. The activated form of MMP-3 enhanced glycerol release as well as TNF-α protein secretion from 3T3-L1 adipocytes. The incubation of adipocytes with MMP-3 inhibited insulin-induced glucose uptake in adipocytes. Furthermore, a high-fat intake increased the expression of MMP-3, decreased the insulin-induced glucose uptake of adipocytes and induced expression of F4/80 in mesenteric fat tissue of C57BL/6 mice.Conclusion:Mφ may cause a pathological link with surrounding adipocytes through the secretion of MMP-3 followed by TNF-α expression in adipocytes in visceral fat tissue.

The Soluble Form of LR11 Protein Is a Regulator of Hypoxia-induced, Urokinase-type Plasminogen Activator Receptor (uPAR)-mediated Adhesion of Immature Hematological Cells

Background: Serum levels of the soluble LR11 fragment (sLR11) increase in patients with acute leukemia. Results: Hypoxia-induced factor (HIF)-1α activation increases LR11 levels, and sLR11 enhances adhesion of HSPCs to BM stromal cells via a uPAR-mediated pathway. Conclusion: sLR11 regulates hypoxia-induced attachment of HSPCs. Significance: sLR11 may stabilize the hematological pool size by controlling HSPC attachment to the BM niche. A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit+ Lin− cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit+ Lin− cells of lr11−/− mice was reduced by hypoxia much more than of lr11+/+ animals. sLR11 induced the adhesion of U937 and c-Kit+ Lin− cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1α, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1α-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1α binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche.