Three founder-related gene mutations (FH Afrikaner-1, -2, and -3) that affect the LDL receptor are responsible for 90% of the familial hypercholesterolemia (FH) in South African Afrikaners. Patients heterozygous for the FH Afrikaner-1 (FH1) mutation, which results in receptors having approximately 20% of normal receptor activity, have significantly lower plasma cholesterol levels and milder clinical symptoms than heterozygotes with the FH Afrikaner-2 mutation, which completely abolishes LDL receptor activity. In this study we re-created the FH3 mutation (Asp154-->Asn) in exon 4 by site-directed mutagenesis and analyzed the expression of the mutant receptors in Chinese hamster ovary cells. The mutation resulted in the formation of LDL receptors that are markedly defective in their ability to bind LDL, whereas binding of apoE-containing beta-VLDL is less affected. The mutant receptors are poorly expressed on the cell surface as a result of significant degradation of receptor precursors. The plasma cholesterol levels of 31 FH3 heterozygotes were similar to FH1 heterozygotes but significantly lower than FH2 heterozygotes. The FH1 and FH3 heterozygotes also tended to be less severely affected clinically (by coronary heart disease and xanthomata) than FH2 patients. This study demonstrates that mutational heterogeneity in the LDL receptor gene influences the phenotypic expression of heterozygous FH and that severity of expression correlates with the activity of the LDL receptor measured in vitro. The results further indicate that knowledge of the specific mutation underlying FH in heterozygotes is valuable in determining the potential risk of premature atherosclerosis and should influence the clinical management of FH patients.

Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized (125)I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. The present study investigated the effects of mmLDL on the expression of endothelin type A (ET(A)) receptors in coronary arteries. Rat coronary arteries were organ-cultured for 24 h. The contractile responses were recorded using a myographic system. ET(A) receptor mRNA and protein expressions were determined using real-time PCR and western blotting, respectively. The results showed that organ-culturing in the presence of mmLDL enhanced the arterial contractility mediated by the ET(A) receptor in a concentration-dependent and time-dependent manner. Culturing with mmLDL (10  μ g/mL) for 24 h shifted the concentration-contractile curves toward the left significantly with increased E(max) of 228% ± 20% from control of 100% ± 10% and significantly increased ET(A) receptor mRNA and protein levels. Inhibition of the protein kinase C, extracellular signal-related kinases 1 and 2 (ERK1/2), or NF- κ B activities significantly attenuated the effects of mmLDL. The c-Jun N-terminal kinase inhibitor or the p38 pathway inhibitor, however, had no such effects. The results indicate that mmLDL upregulates the ETA receptors in rat coronary arterial smooth muscle cells mainly via activating protein kinase C, ERK1/2, and the downstream transcriptional factor, NF- κ B.

Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6Clow monocytes and CD4+ CD25+ FoxP3+ T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

Pain, whether acute or persistent, is a serious medical problem worldwide. However, its management remains unsatisfactory, and new analgesic molecules are required. We show here that TAFA4 reverses inflammatory, postoperative, and spared nerve injury (SNI)-induced mechanical hypersensitivity in male and female mice. TAFA4 requires functional low-density lipoprotein receptor-related proteins (LRPs) because their inhibition by RAP (receptor-associated protein) dose-dependently abolishes its antihypersensitive actions. SNI selectively decreases A-type K+ current (IA) in spinal lamina II outer excitatory interneurons (L-IIo ExINs) and induces a concomitant increase in IA and decrease in hyperpolarization-activated current (Ih) in lamina II inner inhibitory interneurons (L-IIi InhINs). Remarkably, SNI-induced ion current alterations in both IN subtypes were rescued by TAFA4 in an LRP-dependent manner. We provide insights into the mechanism by which TAFA4 reverses injury-induced mechanical hypersensitivity by restoring normal spinal neuron activity and highlight the considerable potential of TAFA4 as a treatment for injury-induced mechanical pain.

Obesity is a major global public health issue involving dyslipidemia, oxidative stress, inflammation, and increased risk of CVD. Weight loss reduces this risk, but the biochemical underpinnings are unclear. We explored how obesity and weight loss after bariatric surgery influence LDL interactions that trigger proatherogenic versus antiatherogenic processes. LDL was isolated from plasma of six patients with severe obesity before (basal) and 6-12 months after bariatric surgery (basal BMI = 42.7 kg/m2; 6-months and 12-months postoperative BMI = 34.1 and 30 kg/m2). Control LDL were from six healthy subjects (BMI = 22.6 kg/m2). LDL binding was quantified by ELISA; LDL size and charge were assessed by chromatography; LDL biochemical composition was determined. Compared to controls, basal LDL showed decreased nonatherogenic binding to LDL receptor, which improved postoperatively. Conversely, basal LDL showed increased binding to scavenger receptors LOX1 and CD36 and to glycosaminoglycans, fibronectin and collagen, which is proatherogenic. One year postoperatively, this binding decreased but remained elevated, consistent with elevated lipid peroxidation. Serum amyloid A and nonesterified fatty acids were elevated in basal and postoperative LDL, indicating obesity-associated inflammation. Aggregated and electronegative LDL remained elevated, suggesting proatherogenic processes. These results suggest that obesity-induced inflammation contributes to harmful LDL alterations that probably increase the risk of CVD. We conclude that in obesity, LDL interactions with cell receptors and extracellular matrix shift in a proatherogenic manner but are partially reversed upon postoperative weight loss. These results help explain why the risk of CVD increases in obesity but decreases upon weight loss.

Diabetic nephropathy is characterized by progressive mesangial expansion. Although we have reported that circulating oxidized LDL-containing immune complexes (oxLDL-IC) are associated with abnormal levels of albuminuria, the underlying mechanisms have not been investigated. In this study, we have studied the effect of oxLDL-IC on collagen IV expression by mesangial cells. We found that oxLDL-IC markedly stimulated collagen IV expression in a concentration- and time-dependent fashion while oxLDL only had moderate effect. We also found that oxLDL-IC stimulated collagen IV expression by engaging Fc gamma receptor (FcγR) I and III, but not FcγRII, and that p38 MAPK, JNK and PKC pathways were involved in collagen IV expression. Furthermore, we found that oxLDL-IC stimulated FcγRI expression, suggesting a positive feedback mechanism involved in oxLDL-IC-stimulated collagen IV expression. Taken together, this study showed that oxLDL-IC stimulated collagen IV in mesangial cells via FcγRI and FcγRIII, and the expression of FcγRI was increased by oxLDL-IC.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-transcriptionally degrades the low density lipoprotein receptors (LDLR). However, it is unknown whether PCSK9 acts directly on the LDLR or if PCSK9 activates another protein that in turn causes degradation of the LDLR.

It is well known that lectin-like oxidized low-density lipoprotein (ox-LDL) and its receptor LOX-1, angiotensin II (AngII) and its type 1 receptor (AT1-R) play an important role in the development of cardiac hypertrophy. However, the molecular mechanism is not clear. In this study, we found that ox-LDL-induced cardiac hypertrophy was suppressed by inhibition of LOX-1 or AT1-R but not by AngII inhibition. These results suggest that the receptors LOX-1 and AT1-R, rather than AngII, play a key role in the role of ox-LDL. The same results were obtained in mice lacking endogenous AngII and their isolated cardiomyocytes. Ox-LDL but not AngII could induce the binding of LOX-1 and AT1-R; inhibition of LOX-1 or AT1-R but not AngII could abolish the binding of these two receptors. Overexpression of wild type LOX-1 with AT1-R enhanced ox-LDL-induced binding of two receptors and phosphorylation of ERKs, however, transfection of LOX-1 dominant negative mutant (lys266ala / lys267ala) or an AT1-R mutant (glu257ala) not only reduced the binding of two receptors but also inhibited the ERKs phosphorylation. Phosphorylation of ERKs induced by ox-LDL in LOX-1 and AT1-R-overexpression cells was abrogated by an inhibitor of Gq protein rather than Jak2, Rac1 or RhoA. Genetically, an AT1-R mutant lacking Gq protein coupling ability inhibited ox-LDL induced ERKs phosphorylation. Furthermore, through bimolecular fluorescence complementation analysis, we confirmed that ox-LDL rather than AngII stimulation induced the direct binding of LOX-1 and AT1-R. We conclude that direct binding of LOX-1 and AT1-R and the activation of downstream Gq protein are important mechanisms of ox-LDL-induced cardiomyocyte hypertrophy.

Low‑density lipoprotein receptors (LDLRs) may serve a role in the diabetogenic effect of statins; however, the effects of statins on LDLR expression and its regulation in the pancreas and islets have yet to be determined. To exclude the long‑term effects of treatment with atorvastatin, which allows mice to adapt, male C57BL/j and apolipoprotein E‑deficient mice were acutely treated with oral atorvastatin for 6 weeks, and glucose homeostasis and LDLR expression in the pancreas and islets were examined. In the present study, it was observed that the short‑term use of atorvastatin affected insulin sensitivity in normal mice and glucose tolerance in hyperlipidemic mice. Furthermore, it was identified that 6 weeks of treatment with atorvastatin suppressed LDLR expression in the pancreas and pancreatic islets in C57BL/j mice, and an increase in proprotein convertase subtilisin/kexin type 9 expression was additionally observed in the pancreas. However, 6 weeks of treatment with atorvastatin did not affect LDLR expression in the pancreas of hyperlipidemic mice. It may be concluded that the short‑term use of atorvastatin disturbs glucose homeostasis and suppresses LDLR expression in the pancreas and pancreatic islets in C57BL/j mice, suggesting that the role of LDLR in the diabetogenic effect of statins requires further investigation.

TNF-α is present in atherosclerotic lesions, activates endothelial adhesion molecule expression, stimulates the release of proinflammatory cytokines and matrix metalloproteinases and promotes smooth muscle cell proliferation and migration. Taken together these observations suggest that TNF-α may be functionally involved in early atherosclerosis development. To further evaluate this hypothesis we compared vascular TNF-α and TNF receptor expression in atherosclerosis-susceptible apoE(-/-)/LDL receptor(-/-) mice and control C57BL/6 mice. The aortas of 8 week old apoE(-/-)/LDLreceptor(-/-) mice displayed immunoreactivity for TNF-α as well as TNF p55 and p75 receptors (2.1 ± 1.6%, 5.6 ± 1.5% and 3.6 ± 1.3% of total media area, respectively), but did not have any detectable lesions. A marginal increase in TNF-α and TNF receptor immunoreactivity was observed at 12 weeks and atherosclerotic plaques were detected in 1 out of 5 animals. At 16 weeks TNF-α expression in the media was increased more than four-fold as compared with 8 week old mice, and atherosclerosis was widespread. TNF-α immunoreactivity was also observed in all plaques. In addition, at the same age a tendency towards increased TNF-α mRNA levels was detected in the double knockout mice compared to age-matched controls. A further increase in TNF-α and TNF receptor immunoreactivity as well as plaque size was observed at 20 weeks. With only a few exceptions, no TNF-α or TNF receptor immunoreactivity was detected in C57BL/6 control mice. These findings demonstrate that medial TNF-α and TNF receptor expression precedes lesion formation in apoE(-/-)/LDL receptor(-/-) mice.

A high level of low-density lipoprotein cholesterol (LDL) is one of the most important risk factors for coronary artery disease (CAD), the leading cause of death worldwide. However, a low concentration of LDL may be protective. Genome-wide association studies revealed that variation in ADTRP gene increased the risk of CAD. In this study, we found that a low concentration of oxidized-LDL induced the expression of ADTRP. Further analyses showed that knockdown of the expression of LDL receptor genes LDLR, CD36, or LOX-1 significantly downregulated ADTRP expression, whereas overexpression of LDLR/CD36/LOX-1 markedly increased ADTRP expression through the NF-κB pathway. Like ADTRP, LDLR, CD36 and LOX-1 were all involved in endothelial cell (EC) functions relevant to the initiation of atherosclerosis. Downregulation of LDLR/CD36/LOX-1 promoted monocyte adhesion to ECs and transendothelial migration of monocytes by increasing expression of ICAM-1, VCAM-1, E-selectin and P-selectin, decreased EC proliferation and migration, and increased EC apoptosis, thereby promoting the initiation of atherosclerosis. Opposite effects were observed with the overexpression of ADTRP and LDLR/CD36/LOX-1 in ECs. Interestingly, through the NF-κB and AKT pathways, overexpression of ADTRP significantly upregulated the expression of LDLR, CD36, and LOX-1, and knockdown of ADTRP expression significantly downregulated the expression of LDLR, CD36, and LOX-1. These data suggest that ADTRP and LDL receptors LDLR/CD36/LOX-1 positively regulate each other, and form a positive regulatory loop that regulates endothelial cell functions, thereby providing a potential protective mechanism against atherosclerosis. Our findings provide a new molecular mechanism by which deregulation of ADTRP and LDLR/CD36/LOX-1 promote the development of atherosclerosis and CAD.

Whole body genetic deletion of AT1a receptors in mice uniformly reduces hypercholesterolemia and angiotensin II-(AngII) induced atherosclerosis and abdominal aortic aneurysms (AAAs). However, the role of AT1a receptor stimulation of principal cell types resident in the arterial wall remains undefined. Therefore, the aim of this study was to determine whether deletion of AT1a receptors in either endothelial cells or smooth muscle cells influences the development of atherosclerosis and AAAs.

Oxidized low-density lipoproteins (oxLDL) and oxLDL-containing immune complexes (oxLDL-IC) contribute to formation of lipid-laden macrophages (foam cells). It has been shown that oxLDL-IC are considerably more efficient than oxLDL in induction of foam cell formation, inflammatory cytokines secretion, and cell survival promotion. Whereas oxLDL is taken up by several scavenger receptors, oxLDL-IC are predominantly internalized through the FCgamma receptor I (FCgamma RI). This study examined differences in intracellular trafficking of lipid and apolipoprotein moieties of oxLDL and oxLDL-IC and the impact on oxidative stress.

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Considerable evidence is available supporting the atherogenic role of immune complexes (IC) formed by modified forms of LDL and their corresponding antibodies in humans and other species. In this study, we assessed the effect of IgG F(ab')2 fragments of murine anti-mouse oxLDL, which binds oxLDL forming IC that cannot interact with Fcγ receptors, on the development of atherosclerosis in diabetic LDL receptor-deficient (LDLR-/-) mice. Immunohistochemical study showed that treatment with the F(ab')2 fragments for 8weeks significantly reduced the content of macrophages and interleukin 6 expression in atherosclerotic lesions. Furthermore, histological study showed that treatment with the same F(ab')2 fragments significantly reduced atherosclerotic lesions in diabetic LDLR-/- mice. Taken together, this study demonstrated for the first time that F(ab')2 fragments of anti-oxLDL IgG inhibited vascular inflammation and atherogenesis in diabetic LDLR-/- mice and uncovered a possible new avenue for therapy in patients at high risk to progress to cardiovascular complications.

The majority of the low-density lipoprotein (LDL) receptors present in the body are expressed in the liver. Therefore, plasma LDL levels significantly correlate with changes in the activity of the hepatic LDL receptor. Based on this, there is a need to understand the regulatory mechanisms that control the hepatic expression of the low-density lipoprotein (LDL) receptor. Herein, we have prepared a functional rat LDL receptor minigene construct that can produce mRNA after splicing. Sequence analysis suggests that this construct has the potential to code for a truncated version of LDL receptor protein. This minigene could be used as a research tool to identify small molecules, natural products, and regulators of the LDL receptor gene that could be developed into LDL receptor-specific activators for therapeutic use.

Electronegative low-density lipoprotein (LDL) (LDL(-)), a modified LDL that is present in blood and exerts atherogenic effects on endothelial cells and monocytes. This study aimed to determine the action of LDL(-) on monocytes differentiated into macrophages. LDL(-) and in vitro-modified LDLs (oxidized, aggregated, and acetylated) were added to macrophages derived from THP1 monocytes over-expressing CD14 (THP1-CD14). Then, cytokine release, cell differentiation, lipid accumulation, and gene expression were measured by ELISA, flow cytometry, thin-layer chromatography, and real-time PCR, respectively. LDL(-) induced more cytokine release in THP1-CD14 macrophages than other modified LDLs. LDL(-) also promoted morphological changes ascribed to differentiated macrophages. The addition of high-density lipoprotein (HDL) and anti-TLR4 counteracted these effects. LDL(-) was highly internalized by macrophages, and it was the major inductor of intracellular lipid accumulation in triglyceride-enriched lipid droplets. In contrast to inflammation, the addition of anti-TLR4 had no effect on lipid accumulation, thus suggesting an uptake pathway alternative to TLR4. In this regard, LDL(-) upregulated the expression of the scavenger receptors CD36 and LOX-1, as well as several genes involved in triglyceride (TG) accumulation. The importance and novelty of the current study is that LDL(-), a physiologically modified LDL, exerted atherogenic effects in macrophages by promoting differentiation, inflammation, and triglyceride-enriched lipid droplets formation in THP1-CD14 macrophages, probably through different receptors.

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

Aberrant activation of inflammation and excess accumulation of lipids play crucial role in the occurrence and progression of atherosclerosis (AS). Quercetin (QCT) has been tested effectively to cure AS. It is widely distributed in plant foods and has been proved to have potential antioxidative and anticancer activities. However, the underlying molecular mechanisms of OCT in AS are not completely understood. In the present study, we stimulated murine RAW264.7 cells with lipopolysaccharide (LPS) or oxidized low-density lipoproteins (ox-LDL) to mimic the development of AS. The data show that QCT treatment leads to an obvious decrease of multiple inflammatory cytokines in transcript level, including interleukin (IL)-1α, IL-1β, IL-2, IL-10, macrophage chemoattractant protein-1 (MCP-1), and cyclooxygenase-2 (COX-2) induced by LPS. Moreover, expressions of other factors that contribute to the AS development, such as matrix metalloproteinase-1 (MMP-1) and suppressor of cytokine signaling 3 (SOCS3) induced by LPS are also downregulated by QCT. Furthermore, we found that QCT suppressed LPS-induced the phosphorylation of STAT3. Meanwhile, QCT could ameliorate lipid deposition and overproduction of reactive oxygen species induced by ox-LDL, and block the expression of lectin-like oxidized LDL receptor-1 (LOX-1) in cultured macrophages. Taken together, our data reveal that QCT has obvious anti-inflammatory and antioxidant virtues and could be a therapeutic agent for the prevention and treatment of AS.