LDL receptor-related protein mediates uptake of aggregated LDL in human vascular smooth muscle cells

Foam cell formation is a key event in the onset and progression of atherosclerotic lesions. We have previously reported that internalization of aggregated low density lipoproteins (agLDLs) by vascular smooth muscle cells (VSMCs) produces cholesteryl ester (CE) accumulation in these cells. The aim of this study was to analyze whether the low density lipoprotein receptor-related protein (LRP) mediates the uptake of agLDL by VSMCs. First, immunocytochemistry and fluorescence microscopic analysis with the use of anti-LRP antibodies indicated that there was a high expression of LRP in VSMCs. Confocal microscopic analysis with the use of agLDLs labeled with fluorochrome 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine and anti-LRP antibodies showed the colocalization of agLDL and LRP. The second approach was to analyze the effect of LRP ligands on agLDL internalization; lactoferrin strongly inhibited CE accumulation from agLDLs (85.0+/-5.7% at 25 microg/mL) by impairing agLDL binding. Coincubation of agLDL with anti-LRP antibodies decreased in a dose-dependent manner agLDL-derived CE accumulation (from 20% at 12.5 microg/mL to 80% at 50 microg/mL). The third approach was to evaluate whether antisense LRP oligodeoxynucleotides were able to block agLDL internalization. Treatment of VSMCs with 5 micromol/L antisense LRP oligodeoxynucleotides reduced agLDL-derived CE accumulation by 84+/-2%. In conclusion, these results from immunologic, biochemical, and molecular interventions demonstrate that LRP mediates the binding and internalization of agLDL in human VSMCs. Because LRP is highly expressed in VSMCs and the uptake of 1 LDL aggregate amounts to the deposition of several hundreds of LDL particles, the uptake of agLDL through LRP could have a crucial role for lipid deposition in VSMCs.     

Angiotensin II upregulates LDL receptor-related protein (LRP1) expression in the vascular wall: a new pro-atherogenic mechanism of hypertension

AIMS: Hypertension is a risk factor for atherothrombotic vascular events. Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, has been associated with the development and progression of atherosclerosis. However, it is not fully known how Ang II contributes to lipid-enriched atherosclerotic lesion formation. In human vascular smooth muscle cells (VSMC), low density lipoprotein (LDL) receptor-related protein (LRP1) internalizes cholesteryl esters (CE) from extracellular matrix-bound aggregated LDL (agLDL). The aim of this study was to investigate the effect of Ang II on LRP1 expression and function in VSMC. METHODS AND RESULTS: Here, we report for the first time that Ang II induces the upregulation of LRP1 expression in VSMC. Ang II (1 microM) induced maximal LRP1 mRNA expression at 12 h and maximal protein overexpression (by 4.10-fold) at 24 h in cultured human VSMC. Ang II effects were functionally translated into an increased CE accumulation from agLDL uptake (by two-fold at 50 microg/mL) that was prevented by the LRP1 ligand lactoferrin and by siRNA-LRP1 treatment. Ang II-LRP1 upregulation and excess CE accumulation from agLDL were prevented by losartan (an AT1 blocker) but not by PD123319 (a specific AT2 blocker). Additionally, in a normolipidaemic rat model, Ang II infusion produced a significant increase in aortic LRP1 expression and lipid infiltration in the arterial intima. CONCLUSION: The in vitro and in vivo data reported here indicate that Ang II upregulates LRP1 receptor expression and LRP1-mediated aggregated LDL uptake in vascular cells.     

Human coronary smooth muscle cells internalize versican-modified LDL through LDL receptor-related protein and LDL receptors

Versican-like proteoglycans are the main component of the intimal extracellular matrix interacting with low density lipoprotein (LDL). The aim of this study has been to investigate the receptors involved in versican-modified LDL uptake by human vascular smooth muscle cells (VSMCs). We have found that versican-LDL interaction leads to the following: (1) monomeric LDL particles that are similar in size and electrophoretic mobility to native LDL but that have a higher capacity to induce intracellular cholesteryl ester (CE) accumulation and (2) fused LDL particles similar in size to those obtained by vortexing. The precipitable fraction of versican-LDL, composed of 50% monomeric and 50% fused LDL particles, induced a dose-response increase in the CE content of VSMCs. Anti-LDL receptor antibody decreased the CE accumulation derived from monomeric LDL particles by 88 +/- 3% and that derived from the total precipitable fraction by 45 +/- 3%. Inhibition of LDL receptor-related protein expression by antisense oligodeoxynucleotides reduced the CE accumulation derived from the precipitable fraction by 65 +/- 2.8%, whereas it did not produce any effect on the CE accumulation derived from monomeric LDL. These results suggest that versican-LDL induces CE accumulation in human VSMCs by the LDL receptor (monomeric particles) and LDL receptor-related protein (fused LDL).     

Mutation in EGFP domain of LDL receptor-related protein 6 impairs cellular LDL clearance

Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6(R611C)) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known. Cholesterol uptake was examined in cells from LRP6(+/-) mice and LRP6(R611C) mutation carriers. Splenic B cells of LRP6(+/-) mice have significantly lower LRP6 expression and low-density lipoprotein (LDL) uptake than those of the wild-type littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6(R611C) mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wild-type receptors show similar affinities for apolipoprotein B at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1 (Niemann-Pick disease type C1), an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6(R611C) was lower compared to wild-type receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apolipoprotein B dissociation from LRP6(R611C) compared to wild-type receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes, which compromises their recycling to the plasma membrane.     

Deletion of macrophage LDL receptor-related protein increases atherogenesis in the mouse

Macrophage low-density lipoprotein receptor-related protein (LRP) mediates internalization of remnant lipoproteins, and it is generally thought that blocking lipoprotein internalization will reduce foam cell formation and atherogenesis. Therefore, our study examined the function of macrophage LRP in atherogenesis. We generated transgenic mice that specifically lack macrophage LRP through Cre/lox recombination. Transplantation of macrophage LRP(-/-) bone marrow into lethally irradiated female LDLR(-/-) recipient mice resulted in a 40% increase in atherosclerosis. The difference in atherosclerosis was not caused by altered serum lipoprotein levels. Furthermore, deletion of macrophage LRP decreased uptake of (125)I-very-low-density lipoprotein compared with wild-type cells in vitro. The increase in atherosclerosis was accompanied by increases in monocyte chemoattractant protein type-1, tumor necrosis factor-alpha, and proximal aorta macrophage cellularity. We also found that deletion of macrophage LRP increases matrix metalloproteinase-9. This increase in matrix metalloproteinase-9 was associated with a higher frequency of breaks in the elastic lamina. Contrary to what was found with other lipoprotein receptors, deletion of LRP increases atherogenesis in hypercholesterolemic mice. Our data support the hypothesis that macrophage LRP modulates atherogenesis through regulation of inflammatory responses.     

Tissue factor induction by aggregated LDL depends on LDL receptor-related protein expression (LRP1) and Rho A translocation in human vascular smooth muscle cells

OBJECTIVE: Low density lipoprotein (LDL) internalized in the vascular wall and modified by binding to extracellular matrix-proteoglycans (ECM) becomes aggregated (agLDL). AgLDL induces tissue factor (TF) expression and activity in human vascular smooth muscle cells (VSMC). TF expression in vascular cells promotes the prothrombotic transformation of the vascular wall. However, the mechanisms by which agLDL induces TF are not known. The aim of this study was to investigate the mechanisms involved in TF activation by extracellular matrix-modified LDL in human VSMC. METHODS AND RESULTS: AgLDL significantly induces TF expression (real time PCR and Western blot analysis) and procoagulant activity (factor Xa generation test) in human VSMC. HMG-CoA reductase inhibition completely prevents agLDL-induced TF expression and partially inhibits agLDL-TF activation. These effects are reverted by geranylgeranyl pyrophosphate (GGPP) but not by farnesyl pyrophosphate (FPP), suggesting the involvement of a geranylated protein in agLDL-TF induction. AgLDL increases Rho A translocation (2-fold) from the cytoplasm to the cell membrane in control but not in simvastatin-treated VSMC. Exoenzyme C3, a specific Rho A inhibitor, completely prevents agLDL-induced TF overexpression and partially agLDL-TF activation. Blocking LRP1, the receptor of agLDL, with anti-LRP1 antibodies or inhibiting LRP1 expression by small interference RNA treatment (siRNA-LRP1) impairs agLDL-induced TF overexpression and activation. CONCLUSIONS: These results demonstrate that TF induction by agLDL depends on LRP1 expression and requires Rho A translocation to the cellular membrane.     

LDL receptor cooperates with LDL receptor-related protein in regulating plasma levels of coagulation factor VIII in vivo

Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII-LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.     

The role of inflammation in atherothrombosis: implications for clinical practice

Inflammation plays a key role in atherothrombosis: in the development of plaques, plaque rupture and thrombus formation. Various biochemical substances have been shown to be involved in the inflammatory process, some with pro-inflammatory activity and others with anti-inflammatory activity. Increased expression of many inflammatory mediators (e.g. C-reactive protein, CD40 ligand, P-selectin and IL-6) has been shown to correlate with increased risk of atherothrombotic events. One possible strategy for primary and secondary prevention is likely to focus on minimizing the inflammatory response and tipping the balance in favour of anti-inflammatory mediators and, therefore, plaque stability.     

Reduced expression of epidermal growth factor receptor-related protein in hepatocellular carcinoma: implications for cancer growth

BACKGROUND/AIMS: The mitogenic signaling pathway of epidermal growth factor receptor (EGFR) is activated in a variety of cancers, including hepatocellular carcinoma (HCC). The recently cloned EGFR-related protein (ERRP) has been proposed to be a negative regulator of EGFR. The expression of ERRP in non-malignant liver cells and in HCC, and its relation to cell proliferation, have not been examined. METHODS: Paraffin sections of formalin-fixed HCC specimens of 51 HCCs and 10 normal liver specimens were immunostained with anti-ERRP and anti-p53 antibodies. To determine the relationship between ERRP expression and cell proliferation, we performed double staining for ERRP and proliferating cell nuclear antigen (PCNA) in the same HCC specimens. RESULTS: In normal liver, ERRP was expressed in 100% of specimens, while in non-malignant liver tissue from HCC, ERRP expression was present in 50% of the specimens (p < 0.001). In contrast, ERRP expression was present only in 14% of the HCC specimens. The expression of ERRP in cancer cells was inversely correlated with proliferative activity and tumor size (p < 0.001, p < 0.05, respectively). No significant correlation was found between p53 expression and the expression of ERRP. CONCLUSIONS: Human HCC, which has been shown to be associated with increased activation of EGFR, exhibits a substantial reduction in ERRP expression. The inverse relationship between ERRP expression, proliferative activity of tumor cells and tumor size suggests that in some HCC, ERRP is a negative regulator of HCC growth.     

Carotid artery atherosclerosis in hypertensive patients with a functional LDL receptor-related protein 6 gene variant

Background and aims

Rare (611C) and common (1062V) variants of the Low-Density Lipoprotein Receptor-Related Protein 6 (LRP6) display reduced activation of Wnt/ß-catenin signaling. The rare gene variant was associated with hypertension, metabolic abnormalities, and early coronary artery disease. We investigated whether the common 1062V LRP6 variant was related to carotid artery atherosclerosis (CAA) in hypertensive patients.

Methods and results

Retrospective study of 334 hypertensive patients (<65 years old) who underwent carotid artery ultrasonography. Hypertension, type 2 diabetes, dyslipidemia, glomerular filtration rate, and smoking habit were evaluated. CAA was defined by the presence of atherosclerotic plaques (focal intima-media thickness ≥1.3 mm). Logistic regression models were used to estimate the independent effect of 1062V allele. The relationship between LRP6 genotypes and LRP6 gene expression in carotid plaques was also investigated. No difference was observed between genotypes in clinical variables except for a slightly higher fasting glucose in 1062V carriers. The 1062V LRP6 variant was an independent risk factor for CAA in both unadjusted (OR 2.08, 95%CI 1.27-3.41, p = 0.003) and adjusted models (OR 1.92, 95%CI 1.09-3.39, p = 0.02). LRP6 was expressed in carotid atherosclerotic plaques at significantly lower levels (p = 0.015) in 1062V carriers.

Conclusion

Beside the role of established risk factors, 1062V variant of LRP6 and CAA are strongly associated in hypertensive patients, making LRP6 a novel relevant candidate gene for atherosclerosis in the presence of hypertension.     

Influence of exonic polymorphisms in the gene for LDL receptor-related protein (LRP) on risk of coronary artery disease

The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional receptor involved in numerous biological processes relevant to vascular biology including lipoprotein metabolism. Several polymorphisms in the LRP gene have been described and in this study we examined their influence on coronary artery disease (CAD). We compared the frequencies of the exon 3 (C766T), exon 6 (C663T), exon 22 (C200T), and four rarer and more recently described polymorphisms in approximately 600 Caucasian subjects aged <50 years with angiographic CAD and approximately 700 similarly aged subjects without symptomatic CAD randomly selected from the community. We found the distribution of exon 22 C200T genotypes to differ significantly between the CAD (CC: 52%, CT: 39%, TT: 9%) and control subjects (CC: 43%, CT: 46%, TT: 11%, P=0.005), with the CC genotype conferring an odds ratio (OR) for CAD of 1.5 (95% CI: 1.2-1.8, P=0.001) despite a lack of significant influence on plasma cholesterol or triglyceride. The other LRP polymorphisms were less common. Two showed an association with CAD; for the exon 3 C766T polymorphism the TT genotype was significantly lower (1.0 vs. 2.7%; OR: 0.36; P=0.04) and, for the exon 6 C663T polymorphism, the heterozygote frequency was higher (6.2 vs. 3.4%; OR: 1.9; P=0.03) in CAD subjects. In conclusion, LRP gene polymorphisms, particularly the relatively common exon 22 C200T polymorphism, are a significant risk factor for premature CAD in Caucasians.     

Sterol regulatory element binding proteins downregulate LDL receptor-related protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by human macrophages

OBJECTIVE: In the extracellular intima, extracellular matrix proteoglycans favor LDL retention and aggregation (agLDL). In contrast to native LDL (nLDL), agLDL induces high intracellular cholesteryl ester (CE) accumulation in macrophages. It has been suggested that LDL receptor-related protein (LRP1) is involved in agLDL binding and internalization by macrophages. The aim of this work was to analyze whether sterol regulatory element binding proteins (SREBPs) modulate LRP1 expression and LRP1-mediated agLDL uptake by human monocyte-derived macrophages (HMDM). METHODS AND RESULTS: The treatment of HMDM with small anti-LRP1 interfering RNA (siRNA-LRP1) led to the specific inhibition of LRP1 mRNA expression and also to the inhibition of LRP1 protein expression in these cells. In siRNA-LRP1-treated HMDM, CE accumulation from agLDL uptake (84.66+/-5 microg CE/mg protein) was reduced by 95.76+/-5.22%. This suggests that LRP1 plays a pivotal role in agLDL uptake by HMDM. N-acetyl-leucyl-leucyl-norleucinal (ALLN), an inhibitor of SREBP catabolism, maintained high levels of active SREBP-2 and SREBP-1 even in the presence of nLDL and agLDL. Therefore, ALLN induced LDL receptor (LDLR) upregulation. Concomitantly, a strong downregulation of LRP1 mRNA and LRP1 protein was observed in ALLN-treated macrophages. By decreasing LRP1 expression levels, ALLN reduced CE accumulation from agLDL at all tested concentrations. CONCLUSIONS: These results suggest that high levels of active SREBPs downregulate LRP1 expression and intracellular CE accumulation in HMDM.     

Decreased hepatic expression of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein in aging rats is associated with delayed clearance of chylomicrons from the circulation

Aging in both humans and rats is associated with the development of insulin resistance and the ensuing alterations in the plasma lipoprotein profile. In this study, young (2 months) and old (15 months) Sprague-Dawley (SD) rats were used to investigate age-related alterations in the chylomicron clearance pathway. Clearance from the blood of an intravenously injected bolus of 14C-labeled cholesterol ester (CE) and 3H-labeled triacylglycerol (TAG) lymph chylomicrons was markedly delayed in the old rats (P < .05). Hepatic expression of the two principal receptors of chylomicron remnant removal, the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP), was determined by ligand blotting and immunoblotting. The old rats expressed 43%+/-7% of the level of LDL receptor in the young animals (P < .05) and 45%+/-16% of the corresponding level of LRP (P < .05). The results suggest that the delayed clearance of chylomicron remnants in this animal model of aging and insulin resistance is due, at least in part, to a decrease in the hepatic expression of LDL receptor and LRP.     

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor

Low-density lipoprotein receptor–related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for C1r and C1s, complement protease mRNA expression was increased in LRP-1–deficient cells, as was expression of inducible nitric oxide synthase (iNOS) and interleukin-6. Regulation of expression of inflammatory mediators was explained by the ability of LRP-1 to suppress basal cell signaling through the IB kinase–nuclear factor-B (NF-B) pathway. LRP-1–deficient macrophages, isolated from mice, demonstrated increased expression of iNOS, C1r, and monocyte chemoattractant protein-1 (MCP-1); MCP-1 expression was inhibited by NF-B antagonism. The mechanism by which LRP-1 inhibits NF-B activity involves down-regulating cell-surface tumor necrosis factor receptor-1 (TNFR1) and thus, inhibition of autocrine TNFR1-initiated cell signaling. TNF-–neutralizing antibody inhibited NF-B activity selectively in LRP-1–deficient cells. We propose that LRP-1 suppresses expression of inflammatory mediators indirectly, by regulating TNFR1-dependent cell signaling through the IB kinase–NF-B pathway.      

Serotonin and the vascular wall

Serotonin possesses both vasoconstrictor and vasodilator properties. The constrictor action of the monoamine can be due to: (a) direct activation of vascular smooth muscle; in most blood vessels, this is mediated by S2-serotonergic receptors; (b) augmentation (amplification) of the action of other endogenous vasoconstrictors such as catecholamines, angiotensin II and the prostanoids; and (c) release of norepinephrine from adrenergic nerves. The dilator action of serotonin can be due to: (a) activation of endothelial cells which release endothelium-derived relaxing factor(s); this response appears to be mediated by S1-serotonergic receptors; (b) direct inhibition of vascular smooth muscle; (c) inhibition of adrenergic neurotransmission by an action on S1-serotonergic prejunctional receptors; and (d) release of other endogenous mediators. The net effect of serotonin on the blood vessel wall depends on: (a) the integrity of the endothelium; (b) the degree of activation of the vascular smooth muscle; (c) the level of sympathetic tone; and (d) local (e.g. PO2, temperature) and chronic (e.g. blood pressure) modulating factors. S2-serotonergic antagonists prevent the constrictor action of serotonin, and often unmask its dilator potential.