Low-density Lipoprotein Receptor-related Protein 1 Levels Research Articles

CART modulates beta-amyloid metabolism-associated enzymes and attenuates memory deficits in APP/PS1 mice

Introduction: Cocaine- and amphetamine-regulated transcript (CART) peptide has been demonstrated to exert neuroprotective effects in stroke and some neurodegeneration diseases. In current study, we investigated the protective effects and underlying mechanisms of CART in APP/PS1 mice.Methods: The protein levels of CART, soluble Aβ1–40 and Aβ1–42 were measured in the hippocampus of APP/PS1 mice by enzyme-linked immunosorbent assay. We determined the mRNA and protein levels of Aβ metabolism-associated enzymes including neprilysin (NEP), insulin-degrading enzyme (IDE), receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP-1) in the hippocampus of APP/PS1 mice using real-time PCR and western blotting. Spatial memory was measured in APP/PS1 mice using the Morris water maze. The phosphorylation of AKT, ERK, p38, and JNK was determined using western blotting.Results: The levels of soluble Aβ1–40 and Aβ1–42 were significantly decreased in the hippocampus of APP/PS1 mice after CART treatment. CART modulated the levels of NEP, IDE, RAGE, and LRP-1. In addition, CART inhibited the MAPK pathways and activated the AKT pathway, whereas inhibition of the AKT pathway decreased the levels of IDE and LRP-1. Furthermore, CART attenuated spatial memory deficits in the APP/PS1 mice.Conclusion: CART decreases the levels of soluble Aβ in the hippocampus of APP/PS1 mice by modulating the expression of Aβ metabolism-associated enzymes, which may be associated with the MAPK and AKT pathways.

Transthyretin stability is critical in assisting beta amyloid clearance- Relevance of transthyretin stabilization in Alzheimer's disease.

The absence of transthyretin (TTR) in AD mice decreases brain Aβ clearance and reduces the low-density lipoprotein receptor-related protein 1 (LRP1). It is possible that neuroprotection by TTR is dependent on its tetramer structural stability, as studies using TTR mutants showed that unstable L55P TTR has low affinity for Aβ, and TTR tetrameric stabilizers such as iododiflunisal ameliorate AD features in vivo. We firstly investigated TTR folding status in human plasma measuring the resistance to urea denaturation. The importance of TTR stability on Aβ internalization was studied in human cerebral microvascular endothelial (hCMEC/D3) and hepatoma cells (HepG2), by flow cytometry. To investigate the fate of Aβ at the blood-brain barrier, Aβ efflux from hCMEC/D3 cells seeded on transwells was measured using ELISA. Further, to assess Aβ colocalization with lysosomes, Lysotracker was used. Moreover, levels of LRP1 were assessed in the liver and plasma of mice with different TTR backgrounds or treated with iododiflunisal. We showed that TTR stability is decreased in AD and that WT TTR and drug-stabilized L55P TTR are able to increase uptake of Aβ.Furthermore, measurement of Aβ efflux showed that stable or stabilized TTR increased Aβ efflux from the basolateral to the apical side. Moreover, HepG2 cells incubated with Aβ in the presence of WT TTR, but not L55P TTR, showed an increased number of lysosomes. Further, in the presence of WT TTR, Aβ peptide colocalized with lysosomes, indicating that only stable TTR assists Aβ internalization, leading to its degradation. Finally, we demonstrated that only stable TTR can increase LRP1 levels. TTR stabilization exerts a positive effect on Aβ clearance and LRP1 levels, suggesting that TTR protective role in AD is dependent on its stability. These results provide relevant information for the design of TTR-based therapeutic strategies for AD.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low‐density lipoprotein receptor‐related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet‐free plasma by enzyme‐linked immunosorbent assay. Plasma‐derived EVs were extracted by size‐exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo‐transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin‐3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9‐ and CD81‐positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin‐3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

FXII promotes proteolytic processing of the LRP1 ectodomain

BackgroundFactor XII (FXII) is a serine protease that is involved in activation of the intrinsic blood coagulation, the kallikrein-kinin system and the complement cascade. Although the binding of FXII to the cell surface has been demonstrated, the consequence of this event for proteolytic processing of membrane-anchored proteins has never been described. MethodsThe effect of FXII on the proteolytic processing of the low-density lipoprotein receptor-related protein 1 (LRP1) ectodomain was tested in human primary lung fibroblasts (hLF), alveolar macrophages (hAM) and in human precision cut lung slices (hPCLS). The identity of generated LRP1 fragments was confirmed by MALDI-TOF-MS. Activity of FXII and gelatinases was measured by S-2302 hydrolysis and zymography, respectively. ResultsHere, we demonstrate a new function of FXII, namely its ability to process LRP1 extracellular domain. Incubation of hLF, hAM, or hPCLS with FXII resulted in the accumulation of LRP1 ectodomain fragments in conditioned media. This effect was independent of metalloproteases and required FXII proteolytic activity. Binding of FXII to hLF surface induced its conversion to FXIIa and protected FXIIa against inactivation by a broad spectrum of serine protease inhibitors. Preincubation of hLF with collagenase I impaired FXII activation and, in consequence, LRP1 cleavage. FXII-triggered LRP1 processing was associated with the accumulation of gelatinases (MMP-2 and MMP-9) in conditioned media. ConclusionsFXII controls LRP1 levels and function at the plasma membrane by modulating processing of its ectodomain. General significanceFXII-dependent proteolytic processing of LRP1 may exacerbate extracellular proteolysis and thus promote pathological tissue remodeling.

Relationship among LRP1 expression, Pyk2 phosphorylation and MMP-9 activation in left ventricular remodelling after myocardial infarction.

Left ventricular (LV) remodelling after myocardial infarction (MI) is a crucial determinant of the clinical course of heart failure. Matrix metalloproteinase (MMP) activation is strongly associated with LV remodelling after MI. Elucidation of plasma membrane receptors related to the activation of specific MMPs is fundamental for treating adverse cardiac remodelling after MI. The aim of current investigation was to explore the potential association between the low‐density lipoprotein receptor‐related protein 1 (LRP1) and MMP‐9 and MMP‐2 spatiotemporal expression after MI. Real‐time PCR and Western blot analyses showed that LRP1 mRNA and protein expression levels, respectively, were significantly increased in peri‐infarct and infarct zones at 10 and 21 days after MI. Confocal microscopy demonstrated high colocalization between LRP1 and the fibroblast marker vimentin, indicating that LRP1 is mostly expressed by cardiac fibroblasts in peri‐infarct and infarct areas. LRP1 also colocalized with proline‐rich tyrosine kinase 2 (pPyk2) and MMP‐9 in cardiac fibroblasts in ischaemic areas at 10 and 21 days after MI. Cell culture experiments revealed that hypoxia increases LRP1, pPyk2 protein levels and MMP‐9 activity in fibroblasts, without significant changes in MMP‐2 activity. MMP‐9 activation by hypoxia requires LRP1 and Pyk2 phosphorylation in fibroblasts. Collectively, our in vivo and in vitro data support a major role of cardiac fibroblast LRP1 levels on MMP‐9 up‐regulation associated with ventricular remodelling after MI.

High glucose facilitated endothelial heparanase transfer to the cardiomyocyte modifies its cell death signature.

The secretion of enzymatically active heparanase (HepA) has been implicated as an essential metabolic adaptation in the heart following diabetes. However, the regulation and function of the enzymatically inactive heparanase (HepL) remain poorly understood. We hypothesized that in response to high glucose (HG) and secretion of HepL from the endothelial cell (EC), HepL uptake and function can protect the cardiomyocyte by modifying its cell death signature. HG promoted both HepL and HepA secretion from microvascular (rat heart micro vessel endothelial cells, RHMEC) and macrovascular (rat aortic endothelial cells, RAOEC) EC. However, only RAOEC were capable of HepL reuptake. This occurred through a low-density lipoprotein receptor-related protein 1 (LRP1) dependent mechanism, as LRP1 inhibition using small interfering RNA (siRNA), receptor-associated protein, or an LRP1 neutralizing antibody significantly reduced uptake. In cardiomyocytes, which have a negligible amount of heparanase gene expression, LRP1 also participated in the uptake of HepL. Exogenous addition of HepL to rat cardiomyocytes produced a dramatically altered expression of apoptosis-related genes, and protection against HG and H2O2 induced cell death. Cardiomyocytes from acutely diabetic rats demonstrated a robust increase in LRP1 expression and levels of heparanase, a pro-survival gene signature, and limited evidence of cell death, observations that were not apparent following chronic and progressive diabetes. Our results highlight EC-to-cardiomyocyte transfer of heparanase to modulate the cardiomyocyte cell death signature. This mechanism was observed in the acutely diabetic heart, and its interruption following chronic diabetes may contribute towards the development of diabetic cardiomyopathy.

Neuronal low-density lipoprotein receptor-related protein 1 (LRP1) enhances the anti-apoptotic effect of intravenous immunoglobulin (IVIg) in ischemic stroke

The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional and multi-ligand endocytic receptor abundantly expressed in neurons. Intravenous immunoglobulin (IVIg) is a purified preparation of plasma-derived human immunoglobulin used for the treatment of several neurological inflammatory disorders, and proposed for the treatment of stroke for its potent neuroprotective effects. LRP1 has been shown to be involved in the transcytosis of IVIg, and IVIg-LRP1 interaction leads to LRP1 tyrosine phosphorylation, which may contribute to the anti-inflammatory effects of IVIg. However, the question remains whether IVIg could induce its neuroprotective effects via LRP1 in neurons under ischemic stroke conditions. In cultured neurons and in a transient ischemic mouse model, ischemia decrease LRP1 levels and phosphorylation, and IVIg blocks these effects. In ischemic neurons, LRP1 antagonism by receptor associated protein (RAP) enhances the activation of pro-death signaling pathways such as nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and caspase-3, and IVIg reduces these effects. When applied to ischemic neuronal cultures, RAP induces a dramatic drop in Akt activation, and IVIg reverses this effect, as it does with the decrease in Bcl-2 levels caused by ischemic injury in the presence of RAP. Altogether, these results show evidence of LRP1 expression and activity modulation by IVIg, and support the role of LRP1 as a partner of IVIg in the execution of its neuroprotective effects.

HIV-1 Tat Regulates Occludin and Aβ Transfer Receptor Expression in Brain Endothelial Cells via Rho/ROCK Signaling Pathway.

HIV-1 transactivator protein (Tat) has been shown to play an important role in HIV-associated neurocognitive disorders. The aim of the present study was to evaluate the relationship between occludin and amyloid-beta (Aβ) transfer receptors in human cerebral microvascular endothelial cells (hCMEC/D3) in the context of HIV-1-related pathology. The protein expressions of occludin, receptor for advanced glycation end products (RAGE), and low-density lipoprotein receptor-related protein 1 (LRP1) in hCMEC/D3 cells were examined using western blotting and immunofluorescent staining. The mRNA levels of occludin, RAGE, and LRP1 were measured using quantitative real-time polymerase chain reaction. HIV-1 Tat at 1 µg/mL and the Rho inhibitor hydroxyfasudil (HF) at 30 µmol/L, with 24 h exposure, had no significant effect on hCMEC/D3 cell viability. Treatment with HIV-1 Tat protein decreased mRNA and protein levels of occludin and LRP1 and upregulated the expression of RAGE; however, these effects were attenuated by HF. These data suggest that the Rho/ROCK signaling pathway is involved in HIV-1 Tat-mediated changes in occludin, RAGE, and LRP1 in hCMEC/D3 cells. HF may have a beneficial influence by protecting the integrity of the blood-brain barrier and the expression of Aβ transfer receptors.

The Matricellular Receptor LRP1 Forms an Interface for Signaling and Endocytosis in Modulation of the Extracellular Tumor Environment.

The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1) has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease inhibitor complexes, and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents. This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

Hypoxia-driven sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) downregulation depends on low-density lipoprotein receptor-related protein 1 (LRP1)-signalling in cardiomyocytes

The maintenance of sarcoplasmic reticulum Ca2+ ATPase (SERCA2) activity is crucial for cardiac function and SERCA2 is dramatically reduced in the heart exposed to hypoxic/ischemic conditions. Previous work from our group showed that hypoxia upregulates the phosphorylated form of the Ca2+-dependent nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (pPyk2) protein levels in a low-density lipoprotein receptor-related protein (LRP1)-dependent manner. Pyk2 in turn may modulate SERCA2 in cardiomyocytes although this remains controversial. We therefore aimed to investigate the role of LRP1 on hypoxia-induced SERCA2 depletion in cardiomyocytes and to establish LRP1 signalling mechanisms involved. Western blot analysis showed that hypoxia reduced SERCA2 concomitantly with a sustained increase in LRP1 and pPyk2 protein levels in HL-1 cardiomyocytes. By impairing hypoxia-induced Pyk2 phosphorylation and HIF-1α accumulation, LRP1 deficiency prevented SERCA2 depletion and reduction of the sarcoplasmic reticulum calcium content in cardiomyocytes. Moreover, the inhibition of Pyk2 phosphorylation (with the Src-family inhibitor PP2) or the specific silencing of Pyk2 (with siRNA-anti Pyk2) preserved low HIF-1α and high SERCA2 levels in HL-1 cardiomyocytes exposed to hypoxia. We determined that the LRP1/Pyk2 axis represses SERCA2 mRNA expression via HIF-1α since HIF-1α overexpression abolished the protective effect of LRP1 deficiency on SERCA2 depletion. Our findings show a crucial role of LRP1/Pyk2/HIF-1α in hypoxia-induced cardiomyocyte SERCA2 downregulation, a pathophysiological process closely associated with heart failure.

Parameters influencing FVIII pharmacokinetics in patients with severe and moderate haemophilia A.

In haemophilia A patients factor VIII (FVIII) recovery and half-life can vary substantially. There are parameters known to modulate FVIII pharmacokinetics (PK), but they explain only about 34% of the variability. The aim of this study was to identify new parameters that influence FVIII PK and thus to expand the current knowledge. FVIII PK were determined in 42 haemophilia A patients (37 severe, 5 moderate) without inhibitor. Patients' characteristics and laboratory parameters were evaluated for an association with FVIII PK. We analysed plasma levels of low-density lipoprotein receptor-related protein 1 (LRP1) and protein C (PC) activity, which had been hypothesized to influence FVIII activity. Furthermore, four variations in intron 6 of the LRP1 gene, which had been shown to influence LRP1, were investigated. FVIII half-life differed widely from 6.2 to 20.7h, with a median of 10.0h. Patients with blood group O had shorter FVIII half-life compared to patients with non-O blood group (median FVIII half-life 9.0h vs. 10.4h, P=0.018). Age was significantly associated with FVIII half-life (r=0.32, P=0.035). Besides age, also VWF antigen (r=0.52, P<0.001) and blood group (r=-0.37, P=0.015) was associated with FVIII half-life. No correlation was found with FVIII- or LRP1-genotype, LRP1 or PC concentrations. Our data showed large differences in FVIII PK between individual patients and revealed age, blood group and VWF levels as important determining factors for FVIII half-life. FVIII genotype or levels of LRP1 or PC had no influence on FVIII PK.

Conditional Knockout of LRP1 in Murine Megakaryocytes and Its Affects on Platelet Factor 4 Biology in Megakaryocytes

We have previously shown that platelet factor 4 (PF4, CXCL4), which is synthesized almost exclusively by megakaryocytes undergoes release intramedullary after which it can undergo reuptake into alpha-granules, but also can be an important negative paracrine regulator of megakaryopoiesis, effecting platelet recovery post-radiation or chemotherapy. Animals that express high levels of human (h) PF4 in addition to their normal levels of murine (m) PF4 (hPF4+) have increased sensitivity to radiation- and chemotherapy-induced thrombocytopenia when compared to wild type (WT) mice or to mice that lack endogenous PF4 (mPF4-/-). Both PF4 reuptake and the negative paracrine effects are at least partially dependent upon the presence of low-density lipoprotein receptor related protein-1 (LRP1) on the surface of megakaryocytes as shown using shRNA suppression of megakaryocyte LRP1 levels. To further understand the role of LRP1 in megakaryopoiesis, we studied LRP1 expressed on primary megakaryocytes in murine models. Homozygous knockout for LRP1 constitutively is embryonically lethal and heterozygous deficiency of LRP1 is insufficient to have an observed effect on PF4 biology. We now established a megakaryocyte-specific knockout of LRP1 using a floxed LRP1 mouse previously described by Rohlman et al, mated to the Cre- PF4 promotor-driven Cre recombinase (Cre+) mice previously described by Tiedt et al. Megakaryocytes from mice that were LRP1fl/fl/Cre+ had no detectable LRP1 mRNA or LRP1 surface protein expression by flow cytometry, while LRP1fl/fl/Cre- mice were essentially identical to WT mice. Baseline platelet counts in LRP1fl/fl/Cre+ and LRP1fl/fl/Cre- mice did not different from each other, and there was no difference in bone marrow derived megakaryocyte ploidy. PF4 available in platelet releasate of LRP1fl/fl/Cre+ platelets was also significantly less than in LRP1fl/fl/Cre- platelets (208 ± 42 vs. 362 ± 47 IU/106 platelets, p=0.002) consistent with a role of LRP1 in PF4 reuptake into megakaryocytes in the steady-state and demonstrating that >42% of PF4 may be released during PF4 megakaryopoiesis and requires megakaryocyte LRP1 expression. In siru cultured LRP1fl/fl/Cre+ megakaryocytes exposed to exogenous hPF4 has a lower level of total PF4 levels than LRP1fl/fl/Cre- megakaryocytes (191 ± 7 vs. 236 ± 17 IU/106 cells, respectively (p=0.03)). A similar effect was seen in liquid bone marrow culture assays. Finally, while LRP1fl/fl/Cre+/hPF4+ mice had similar platelet count recovery after irradiation compared to LRP1fl/fl/Cre+/WT mice, treatment of these mice with a heparin-derivative (ODSH) shown to significantly improve platelet count recovery and animal survival in both WT and hPF4+ mice had no effect on either platelet count recovery or animal survival in animals that were also LRP1fl/fl/Cre+. These data demonstrate that nearly half of the total PF4 in megakaryocytes undergoes recycling in vivo and that LRP1 is important for this phenomenon in the steady-state. LRP1 is also important in the negative paracrine effect of PF4 in stress megakaryopoiesis though LRP1 may affect megakaryocyte biology by non-PF4-dependent pathways as well. Whether the two observations – PF4 uptake and negative paracrine effects – are mechanistically related or are distinct LRP1-dependent pathways now needs to be elucidated. DisclosuresXiao:ECRI Institute: Employment.

The LRP1-independent mechanism of PAI-1-inudced migration in CpG-ODN activated macrophages

CpG-oligodeoxynucleotides (CpG-ODNs) induces plasminogen activator inhibitor type-1 (PAI-1) expression in macrophages, leading to enhanced migration through vitronectin. However, the precise role of low-density lipoprotein receptor-related protein 1 (LRP1) in PAI-1 induced migration of macrophages in the inflammatory environment is not known. In this study, we elucidated a novel mechanism describing the altered role of LRP1 in macrophage migration depending on the activation state of the cells. Experimental evidence clearly shows that the blocking of LRP1 function inhibited the PAI-induced migration of resting RAW 264.7 cells through vitronectin but exerted a pro-migratory effect on CpG-ODN-activated cells. We also demonstrate that CpG-ODN downregulates the protein and mRNA levels of LRP1 both in vivo and in vitro, a function that depends on the NF-κB signaling pathway, resulting in reduced internalization of PAI-1. This work illustrates the distinct mechanism that PAI-1-induced migration of CpG-ODN-activated cells through vitronectin depends on the interaction of PAI-1 with vitronectin but not LRP1 unlike in the resting cells, where the migration is LRP1 dependent and vitronectin independent. In conclusion, our experimental results demonstrate the altered function of LRP1 in the migration of resting and activated macrophages in the context of microenvironmental extracellular matrix components.

Sevoflurane alters the expression of receptors and enzymes involved in Aβ clearance in rats

Patients with Alzheimer's disease (AD) exhibit a failure in the clearance of amyloid β peptides (Aβ) from the central nervous system. Previous studies have suggested an association between anesthesia and the occurrence of AD. The aim of the present report was to further explore this possibility. Animals were administered sevoflurane for 2 h. We performed immunohistochemistry and real-time polymerase chain reaction to assess the levels of low-density lipoprotein receptor-related protein 1 (LRP-1), the receptor for advanced glycation end products (RAGE) protein, insulin-degrading enzyme (IDE), and neprilysin (NEP) in aged and young rat's brain. Levels of LRP-1 were significantly decreased, while those of RAGE increased in the aged and young groups. Immunoreactivity for IDE was significantly decreased at 3 and increased at 15 days in the young group. In contrast, immunoreactivity for NEP was significantly increased at 1 but decreased at 15 days in aged rats. Levels of IDE messenger RNA (mRNA) were significantly decreased at 3 and 7 days in the aged group but was consistently decreased at 1, 3, 7, and 15 days in the young group. Levels of NEP mRNA were significantly decreased in the aged group but increased in the young group at 1, 3, 7, and 15 days. Sevoflurane leads to a reduction in the levels of LRP-1, while increasing RAGE and decreasing IDE and NEP in both aged and, to a lesser extent, young rat's brain. These receptor and enzymatic changes may promote the accumulation of Aβ in brain tissues and thus exacerbate Alzheimer's-like pathology.

Exploration of effects of emodin in selected cancer cell lines: enhanced growth inhibition by ascorbic acid and regulation of LRP1 and AR under hypoxia‐like conditions

This study explores the link between the antiproliferative activity of emodin through the generation of reactive oxygen species (ROS) in various cancer cell lines and the expression of the androgen receptor (AR) in the prostate cancer cell lines LNCaP (androgen-sensitive) and PC-3 (androgen-refractory), as well as the pro-metastatic low-density lipoprotein receptor-related protein 1 (LRP1) in the above prostate cancer cells and the nonprostate cell lines A549 (lung), HCT-15 (colon) and MG-63 (bone) under normoxic and hypoxia-like conditions. Among all cell lines, emodin showed most growth inhibition in LNCaP, followed by A549. The mechanism of cytotoxicity of emodin was postulated to be the widely reported ROS generation, based on the observations of poor in vitro radical-scavenging activity and increased growth inhibition of emodin by ascorbic acid (AA) pre-treatment owing to the additive effects of ROS generation by emodin and pro-oxidant effects of AA. Emodin downregulated AR in LNCaP under normoxic and hypoxia-like conditions (simulated by CoCl(2)) and LRP1 under normoxia. Emodin upregulated LRP1 in other cell lines, except HCT-15, under normoxic, and even more markedly under hypoxia-like conditions. The downregulation of AR in LNCaP and upregulation of LRP1 in all cell lines, except HCT-15, under hypoxia-like conditions along with growth inhibition by emodin, suggests that emodin may be a useful therapeutic option against androgen-sensitive prostate cancer and other such LRP1-expressing cancers to attempt the targeting of the elevated LRP1 levels to allow the uptake of emodin and/or any other accompanying therapeutic agents by LRP1.

Evaluation for Antithrombotic Agents by a Thrombotic Model in Animals

To investigate cerebral infarction and intracranial hemorrhage (ICH) in relation to antithrombotic agents, we established an animal stroke model induced by using a combination of a photosensitive dye and local green photoillumination. Microplasmin (μPli), a derivative of plasmin lacking the five "kringle" domains, was administered in this model, and its effect was studied using magnetic resonance imaging. μPli treatment reduced cerebral damage 24 h after middle cerebral artery occlusion; it also reduced the expansion of the positive area on perfusion-weighted images between 1 and 24 h and the degree of neurological deficits. Tissue-type plasminogen activator (t-PA), a serine proteinase that converts plasminogen to plasmin, has been approved for treating acute ischemic stroke, but delayed treatment is associated with increased risk of ICH. Plasmin participates in the degradation of fibrin, causing clot lysis, and of various extracellular matrix proteins, either directly or via the activation of matrix metalloproteinases (MMPs). In this study, we observed that MMP-3 is relatively important in the enhanced risk of ICH induced by delayed t-PA treatment for ischemic stroke. In particular, the binding of t-PA with low-density lipoprotein receptor-related protein (LRP) results in the release of MMP-3 by endothelial cells. LRP production is upregulated in endothelial cells exposed to ischemia, and elevated LRP levels have been implicated in the increased ICH risk associated with delayed t-PA treatment. This implies that the t-PA/LRP/MMP-3 pathway may be a suitable target for developing strategies to improve the therapeutic efficacy of t-PA in acute ischemic stroke.

LRP1 expression in cerebral cortex, choroid plexus and meningeal blood vessels: Relationship to cerebral amyloid angiopathy and APOE status

APOE genotype is a risk factor for Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). The risk and severity of CAA increase with possession of APOE ɛ4, whereas APOE ɛ2 increases the risk of vessel rupture. Uptake of Aβ by cerebrovascular smooth muscle cells (CVSMCs) is mediated by low-density lipoprotein receptor-related protein-1 (LRP1). To determine whether APOE influences CAA by altering LRP1 expression, particularly by CVSMCs, we analysed APOE genotype, CAA severity, and LRP1 levels in post-mortem cerebral cortex, choroid plexus and meningeal vessels. LRP1 mRNA and protein were not related to CAA severity and presence. LRP1 mRNA was increased in meningeal vessels, but not cortex or choroid plexus, in AD and in association with APOE ɛ4, and was decreased in association with APOE ɛ3. In brains with CAA, APOE ɛ2 was associated with decreased LRP1 protein in meningeal vessels, and ɛ3 with increased LRP1 in choroid plexus. These findings suggest that APOE may influence the severity of CAA through altered expression of LRP1.

Aβ1-40 clearance from the brain by regulation of LRP-1: A study on effects of insulin, rosiglitazone or the combination in type 2 diabetic GK rats

BackgroundThere is high incidence of cerebral amyloid beta (Aβ) deposition in insulin-resistant type 2 diabetes mellitus. Low-density lipoprotein receptor-related protein 1 (LRP-1) mediates the 40-amino-acid form of Aβ (Aβ1-40) efflux from brain to periphery. In LRP-1-mediated Aβ regulation, insulin and peroxisome proliferator-activated receptor-γ (PPARγ) were found to have important effect on the expression and function of LRP-1. We therefore hypothesised that insulin and PPARγ-agonist (rosiglitazone), alone or in combination may affect LRP-1 distribution and expression, and then reduce Aβ1-40 deposition in diabetes brain. MethodsGoto-Kakizaki (GK) rats were used as type 2 diabetic rats model. The expression of LRP-1 and Aβ1-40 in brain microvessels was detected by immunohistochemistry. The expression levels of LRP-1 and Aβ1-40 in brain tissue were estimated by enzyme-linked immunosorbent assays (ELISA). LRP-1mRNA was detected by RT-PCR. ResultsWith diabetes mellitus progression, Aβ1-40 deposition in brain tissue increased obviously. Administration of rosiglitazone to GK rats caused significant increase in the LRP-1 protein content and specific mRNA level of this receptor with reduction of levels of Aβ1-40 in GK brain tissue when compared to GK group. Also, there was a significant increase in the LRP-1 protein content in plasma membrane fraction of brain tissue, whereas the expression in the whole lysate was not affected with a significant reduction of levels of Aβ1-40 in GK brain by insulin treatment. Compared to GK with insulin group, there was a great increase in the LRP-1 protein content in the brain in the group administered a combination of insulin with rosiglitazone. ConclusionsInsulin or rosiglitazone could decrease accumulation of Aβ1-40 in the GK brain which appeared to be mediate, at least in part, via LRP-1 and in combination use, rosiglitazone seemed to be able to sensitize/enhance insulin's inducing the subcellular translocation of LRP-1 to the plasma membrane. In this context, it is probably beneficial to combine insulin with rosiglitazone to benefit type 2 diabetes mellitus based on the known LRP1-mediated anti-Aβ1-40 effect.

Low Density Lipoprotein Receptor-Related Protein 1 Dependent Endosomal Trapping and Recycling of Apolipoprotein E

BackgroundLipoprotein receptors from the low density lipoprotein (LDL) receptor family are multifunctional membrane proteins which can efficiently mediate endocytosis and thereby facilitate lipoprotein clearance from the plasma. The biggest member of this family, the LDL receptor-related protein 1 (LRP1), facilitates the hepatic uptake of triglyceride-rich lipoproteins (TRL) via interaction with apolipoprotein E (apoE). In contrast to the classical LDL degradation pathway, TRL disintegrate in peripheral endosomes, and core lipids and apoB are targeted along the endocytic pathway for lysosomal degradation. Notably, TRL-derived apoE remains within recycling endosomes and is then mobilized by high density lipoproteins (HDL) for re-secretion. The aim of this study is to investigate the involvement of LRP1 in the regulation of apoE recycling.Principal FindingsImmunofluorescence studies indicate the LRP1-dependent trapping of apoE in EEA1-positive endosomes in human hepatoma cells. This processing is distinct from other LRP1 ligands such as RAP which is efficiently targeted to lysosomal compartments. Upon stimulation of HDL-induced recycling, apoE is released from LRP1-positive endosomes but is targeted to another, distinct population of early endosomes that contain HDL, but not LRP1. For subsequent analysis of the recycling capacity, we expressed the full-length human LRP1 and used an RNA interference approach to manipulate the expression levels of LRP1. In support of LRP1 determining the intracellular fate of apoE, overexpression of LRP1 significantly stimulated HDL-induced apoE recycling. Vice versa LRP1 knockdown in HEK293 cells and primary hepatocytes strongly reduced the efficiency of HDL to stimulate apoE secretion.ConclusionWe conclude that LRP1 enables apoE to accumulate in an early endosomal recycling compartment that serves as a pool for the intracellular formation and subsequent re-secretion of apoE-enriched HDL particles.

CDP-choline treatment induces brain plasticity markers expression in experimental animal stroke

We investigated the effect of CDP-choline on brain plasticity markers expression in the acute phase of cerebral infarct in an experimental animal model. Male Sprague–Dawley rats were subjected to permanent middle cerebral artery occlusion (pMCAO) and treated or not with CDP-choline (500mg/kg) daily for 14days starting 30min after pMCAO. Functional status was evaluated with Roger’s test; lesion volume with magnetic resonance imaging (MRI) and hematoxylin and eosin staining (H&E); cell death with TUNEL; cellular proliferation with BrdU immunohistochemistry; vascular endothelial growth factor (VEGF), synaptophysin, glial fibrillary acidic protein (GFAP) and low-density lipoprotein receptor-related protein (LRP) by immunofluorescence and Western-blot techniques. CDP-choline significantly improved functional recovery and decreased lesion volume on MRI, TUNEL-positive cell number and LRP levels at 14days. In addition, CDP-choline significantly increased BrdU, VEGF and synaptophysin values and decreased GFAP levels in the peri-infarct zone compared with the infarct group. In conclusion, our data indicate that CDP-choline improved functional recovery after permanent middle cerebral artery occlusion in association with reductions in lesion volume, cell death and LRP expression. In fact, CDP-choline increased cell proliferation, vasculogenesis and synaptophysin levels and reduced GFAP levels in the peri-infarct area of the ischemic stroke.