The effects of Danggui-Shaoyao-San on neuronal degeneration and amyloidosis in mouse and its molecular mechanism for the treatment of Alzheimer's disease.

The abnormal deposition of the extracellular amyloid-ß peptide is the typical pathological hallmark of Alzheimer's disease. Strategies to reduce the amyloid-ß deposition effectively alleviate the neuronal degeneration and cognitive deficits of Alzheimer's disease. Danggui-Shaoyao-San has been considered a useful therapeutic agent known for the treatment of Alzheimer's disease. However, the mechanism of Danggui-Shaoyao-San for the treatment of Alzheimer's disease remains unclear. We investigated Danggui-Shaoyao-San's effect on amyloidosis and neuronal degeneration in an APP/PS1 mouse model. We found Danggui-Shaoyao-San alleviated the cognitive deficits in APP/PS1 mice. Additionally, Danggui-Shaoyao-San ameliorated the neuronal degeneration in these mice. Danggui-Shaoyao-San reduced the amyloidosis and amyloid-ß1-42 deposition in APP/PS1 mouse brain and down-regulated the receptor for advanced glycation end products, and up-regulated the level of low-density lipoprotein receptor-related protein-1. However, the protein expression of the ß-amyloid precursor protein, ß-Secretase and presenilin-1 (PS1) in the amyloid-ß production pathway, and the expression of neprilysin and insulin-degrading enzyme in the amyloid-ß degradation pathway were not altered. Our findings collectively suggest that Danggui-Shaoyao-San could ameliorate the amyloidosis and neuronal degeneration of Alzheimer's disease, which may be associated with its up-regulation lipoprotein receptor-related protein-1 and down-regulation of the receptor for advanced glycation end products.

Deferoxamine therapy reduces brain hemin accumulation after intracerebral hemorrhage in piglets.

Hemopexin (Hpx) is critical for hemin scavenging after the erythrocyte lysis that occurs following intracerebral hemorrhage (ICH). Low-density lipoprotein receptor-related protein-1 (LRP1, also called CD91) is an important receptor through which the hemin-Hpx complex can undergo endocytosis. This study investigated changes in the hemin-Hpx-CD91 axis in both hematoma and perihematomal tissue in a large animal ICH model. The effect of deferoxamine (DFX) on hemin-Hpx-CD91 was also examined. The study consisted of two parts. First, piglets had an injection of autologous blood into the right frontal lobe of brain and were euthanized from day 1 to day 7. Hematoma and perihematomal tissue of brains were used for hemin assay, immunohistochemistry, and immunofluorescence. Second, piglets with ICH were treated with deferoxamine or vehicle, and were euthanized for hemin measurement and Hpx and CD91 immunohistochemistry. We found that there was an increase of hemin levels within the hematoma and perihematomal brain tissue after ICH. Hpx and CD91-positive cells were present in the clot and perihematomal tissue from day 1. Hpx and CD91 positive cells were Iba1 positive. After DFX therapy, hemin dropped markedly in the hematoma and perihematomal brain tissue. Furthermore, DFX treatment decreased the number of Hpx and CD91 positive cells in and around the hematoma. In conclusion, hemin accumulation occurs in and around the hematoma. Increases in Hpx and CD91 may be important in scavenging that hemin. DFX treatment decreased hemin release from the hematoma and reduced the expression of Hpx and CD91.

Matriptase Induction of Metalloproteinase-Dependent Aggrecanolysis In Vitro and In Vivo: Promotion of Osteoarthritic Cartilage Damage by Multiple Mechanisms.

OBJECTIVE: To assess the ability of matriptase, a type II transmembrane serine proteinase, to promote aggrecan loss from the cartilage of patients with osteoarthritis (OA) and to determine whether its inhibition can prevent aggrecan loss and cartilage damage in experimental OA. METHODS: Aggrecan release from human OA cartilage explants and human stem cell-derived cartilage discs was evaluated, and cartilage-conditioned media were used for Western blotting. Gene expression was analyzed by real-time polymerase chain reaction. Murine OA was induced by surgical destabilization of the medial meniscus, and matriptase inhibitors were administered via osmotic minipump or intraarticular injection. Cartilage damage was scored histologically and aggrecan cleavage was visualized immunohistochemically using specific neoepitope antibodies. RESULTS: The addition of soluble recombinant matriptase promoted a time-dependent release of aggrecan (and collagen) from OA cartilage, which was sensitive to metalloproteinase inhibition and protease-activated receptor 2 antagonism. Although engineered human (normal) cartilage discs failed to release aggrecan following matriptase addition, both matrix metalloproteinase- and aggrecanase-mediated cleavages of aggrecan were detected in human OA cartilage. Additionally, while matriptase did not directly degrade aggrecan, it promoted the accumulation of low-density lipoprotein receptor-related protein 1 (LRP-1) in conditioned media of the OA cartilage explants. Matriptase inhibition via neutralizing antibody or small molecule inhibitor significantly reduced cartilage damage scores in murine OA, which was associated with reduced generation of metalloproteinase-mediated aggrecan cleavage. CONCLUSION: Matriptase potently induces the release of metalloproteinase-generated aggrecan fragments as well as soluble LRP-1 from OA cartilage. Therapeutic targeting of matriptase proteolytic activity reduces metalloproteinase activity, further suggesting that this serine proteinase may have potential as a disease-modifying therapy in OA.

Inhibition of Shedding of Low-Density Lipoprotein Receptor-Related Protein 1 Reverses Cartilage Matrix Degradation in Osteoarthritis.

OBJECTIVE: The aggrecanase ADAMTS-5 and the collagenase matrix metalloproteinase 13 (MMP-13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP-1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP-1. The aim of this study was to identify the LRP-1 sheddase(s) in human cartilage and to test whether inhibition of LRP-1 shedding prevents cartilage degradation in OA. METHODS: Cell-associated LRP-1 and soluble LRP-1 (sLRP-1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP-1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively. RESULTS: Shedding of LRP-1 was increased in OA cartilage compared with normal tissue. Shed sLRP-1 bound to ADAMTS-5 and MMP-13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane-bound metalloproteinases, ADAM-17 and MMP-14, were identified as the LRP-1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage. CONCLUSION: Shedding of LRP-1 is a key link to OA progression. Local inhibition of LRP-1 sheddase activities of ADAM-17 and MMP-14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach.

Imatinib mesylate stimulates low-density lipoprotein receptor-related protein 1-mediated ERK phosphorylation in insulin-producing cells.

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and multi-functional type I cell surface membrane protein, which is known to be phosphorylated by the activated platelet-derived growth factor receptor (PDGFR). The tyrosine kinase inhibitor imatinib, which inhibits PDGFR and c-Abl, and which has previously been reported to counteract ß-cell death and diabetes, has been suggested to reduce atherosclerosis by inhibiting PDGFR-induced LRP1 phosphorylation. The aim of the present study was to study LRP1 function in ß-cells and to what extent imatinib modulates LRP1 activity. LRP1 and c-Abl gene knockdown was performed by RNAi using rat INS-1 832/13 and human EndoC1-ßH1 cells. LRP1 was also antagonized by treatment with the antagonist low-density lipoprotein receptor-related protein associated protein 1 (LRPAP1). We have used PDGF-BB, a PDGFR agonist, and apolipoprotein E (ApoE), an LRP1 agonist, to stimulate the activities of PDGFR and LRP1 respectively. Knockdown or inhibition of LRP1 resulted in increased hydrogen peroxide (H2O2)- or cytokine-induced cell death, and glucose-induced insulin release was lowered in LRP1-silenced cells. These results indicate that LRP1 function is necessary for ß-cell function and that LRP1 is adversely affected by challenges to ß-cell health. PDGF-BB, or the combination of PDGF-BB+ApoE, induced phosphorylation of extracellular-signal-regulated kinase (ERK), Akt and LRP1. LRP1 silencing blocked this event. Imatinib blocked phosphorylation of LRP1 by PDGFR activation but induced phosphorylation of ERK. LRP1 silencing blocked imatinib-induced phosphorylation of ERK. Sunitinib also blocked LRP1 phosphorylation in response to PDGF-BB and induced phosphorylation of ERK, but this latter event was not affected by LRP1 knockdown. siRNA-mediated knockdown of the imatinib target c-Abl resulted in an increased ERK phosphorylation at basal conditions, with no further increase in response to imatinib. Imatinib-induced cell survival of tunicamycin-treated cells was partially mediated by ERK activation. We have concluded that imatinib promotes LRP1-dependent ERK activation, possibly via inhibition of c-Abl, and that this could contribute to the pro-survival effects of imatinib on ß-cells.

The hypolipidemic effect of cilostazol can be mediated by regulation of hepatic low-density lipoprotein receptor-related protein 1 (LRP1) expression.

OBJECTIVES: Cilostazol, a selective phosphodiesterase 3 (PDE3) inhibitor, is a vasodilator and an anti-thrombotic agent. The mechanism whereby cilostazol reduces plasma triglyceride is not completely understood. Here we investigated the effect of cilostazol on a remnant lipoprotein receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which has been reported to play an essential role in clearance of circulating triglyceride in the liver. MATERIALS/METHODS: Total cellular expression, and functional and transcriptional regulation of LRP1 were analyzed in human hepatocarcinoma cell lines incubated with cilostazol. Also, C57BL/6 mice were subjected to high-fat diet (60% kcal) and cilostazol (30 mg/kg) treatment for 10 weeks. RESULTS: Cilostazol increased both mRNA and protein expression of LRP1 in HepG2 and Hep3B cells. In addition, enhanced transcriptional activity of the LRP1 promoter containing a peroxisome proliferator response element (PPRE) was observed after cilostazol exposure. Cilostazol treatment enhanced the uptake of lipidated apoE3, and this effect was abolished when LRP1 was silenced by siRNA knockdown. High-fat diet induced hyperglycemia with high level of plasma triglycerides, and reduced hepatic LRP1 expression in mice. Treatment with cilostazol for the same period of time, however, successfully prevented this down-regulation of LRP1 expression and reduced plasma triglycerides. CONCLUSION: Taken together, our results demonstrated that cilostazol enhances LRP1 expression in liver by activating PPARγ through the PPRE in the LRP1 promoter. Increased hepatic LRP1 may be essential for the reduction of circulating triglycerides brought about by cilostazol.

Smooth muscle LDL receptor-related protein-1 inactivation reduces vascular reactivity and promotes injury-induced neointima formation.

OBJECTIVE: Defective smooth muscle expression of LDL receptor-related protein-1 (Lrp1) increases atherosclerosis in hypercholesterolemic mice. This study explored the importance of smooth muscle Lrp1 expression under normolipidemic conditions. METHODS AND RESULTS: Smooth muscle cells isolated from control (smLrp1(+/+)) and smooth muscle-specific Lrp1 knockout (smLrp1(-/-)) mice were characterized based on morphology, smooth muscle marker protein expression levels, and growth rates in vitro. Vascular functions were assessed by aortic constrictive response to agonist stimulation in situ and neointimal hyperplasia to carotid arterial injury in vivo. The smLrp1(-/-) smooth muscle cells displayed reduced alpha-actin and calponin expression and an accelerated growth rate attribtuable to sustained phosphorylation of platelet-derived growth factor receptor (PRGFR) and protein kinase B/Akt. Vasoconstrictive response to agonist stimulation was impaired in aortic rings isolated from smLrp1(-/-) mice. Injury-induced neointimal hyperplasia was significantly increased in smLrp1(-/-) mice. The increase in neointima was associated with corresponding elevated activation of PDGFR signaling pathway. CONCLUSIONS: Smooth muscle expression of Lrp1 is important in maintaining normal vascular functions under normolipidemic conditions. The absence of Lrp1 expression results in greater smooth muscle cell proliferation, deficient contractile protein expression, impairment of vascular contractility, and promotion of denudation-induced neointimal hyperplasia.

[Multiple involvements of LRP-1 receptor in tumor progression]. / Implications multiples du récepteur LRP-1 dans la progression tumorale.

Extensive proteolytic remodeling processes constitute a critical step during tumor progression. The endocytic receptor low-density lipoprotein receptor-related protein-1 (LRP-1), by its function in the clearance of multiple extracellular proteases involved in metastatic spreading, has long been considered as a putative tumor suppressor. Moreover, the receptor is likely to control the peritumoral microenvironment by internalization of growth factors and matricial proteins and could therefore participate to the control of signaling events involved in survival and proliferation of cancer cells. Nevertheless, recent data lead to reconsider the initially attributed antitumor properties of LRP-1. A more complex model seems to emerge in which LRP-1 could constitute a sensor of pericellular environment and regulate the membrane proteome dynamics. By its control of focal adhesions composition and turn-over, regulation of the cytoskeleton organization and integrin endocytic recycling, LRP-1 appears as a crucial actor of the epithelial-mesenchymal transition, thereby reinforcing the aggressive phenotype of malignant cells. LRP-1 partitioning into rafts and association with tissue-type and tumor grade specific intracellular scaffold proteins appear crucial to determine its function in tumor progression. Those emerging aspects present numerous promising perspectives in oncology and allow envisaging the development of innovative strategies of control of tumor progression through the targeting of LRP-1.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a relevant target for diabetic vasculopathy?

Mechanisms through which major risk factors accelerate diabetic angiopathy include low density lipoprotein (LDL) oxidation and advanced glycation end products (AGEs) formation. Lectin-like oxidized LDL receptor (LOX-1) is a newly identified vascular receptor for oxidized LDL (oxLDL) and AGEs. LOX-1 is up-regulated in vascular endothelium of diabetic animals and thus may be relevant to the development and progression of human diabetic vasculopathy. The mechanisms responsible for LOX-1 induction in diabetes remain unclear but appear to involve metabolic and inflammatory stimuli relevant to diabetes. Such factors may impact on LOX-1-mediated pro-atherogenic events, including endothelial dysfunction and plaque destabilization. Previous studies have shown that drugs commonly used in the treatment of type 2 diabetic patients, including statins and antidiabetic agents, inhibit endothelial LOX-1 expression. This review summarizes recent advances related to the role of LOX-1 in macrovascular diseases, its regulation by some derangements commonly found in diabetic patients and its modulation by vasculoprotective drugs.

Kallikrein gene 'knock-down' by small interfering RNA transfection induces a profibrotic phenotype in rat mesangial cells.

BACKGROUND: Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by 'knocking down' its expression using specific small interfering RNAs (siRNA). METHODS: Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed. RESULTS: Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein-kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-beta protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance. CONCLUSION: These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.

Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes.

Soluble amyloid-beta peptide (Abeta) exists in the form of monomers and oligomers, and as complexes with Abeta-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Abeta(1-40) [hAbeta(1-40)] from the rat brain across the blood-brain barrier. Incubation of [(125)I]hAbeta(1-40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [(125)I]hAbeta(1-40) dimer was significantly decreased by 92.7% compared with that of [(125)I]hAbeta(1-40) monomer. Pre-incubation with LRP-1 ligands, such as activated alpha2-macroglobulin (alpha2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [(125)I]hAbeta(1-40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [(125)I]hAbeta(1-40) monomer elimination. Purified [(125)I]hAbeta(1-40)/activated alpha2M complex and [(125)I]activated alpha2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [(125)I]activated alpha2M, and enhancement of [(125)I]hAbeta(1-40) uptake upon pre-incubation with apoE, suggesting that [(125)I]activated alpha2M and [(125)I]hAbeta(1-40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAbeta(1-40) from the brain across the blood-brain barrier.

Estradiol replacement increases the low-density lipoprotein receptor related protein (LRP) in the mouse brain.

Numerous epidemiology studies have shown protective effects of hormone therapy (HT) on chronic neurological diseases. We have proposed that some of the neuroprotective effects of estrogen are mediated by apolipoprotein E (apoE). Polymorphisms of receptors for apoE modify the risk for dementia. To our knowledge, no reports exist showing CNS effects of estrogen replacement on members of the low-density lipoprotein receptor family. The current study focused on the effect of estradiol-17beta (E2) replacement on protein expression of two members of the receptor family, the low-density lipoprotein receptor (LDL-r) and low-density lipoprotein receptor related protein (LRP) in ovariectomized mice. Five days of E2 replacement significantly increased LRP expression in the hippocampus, olfactory bulb and neocortex but not in cerebellum. In contrast, E2 treatment decreased LDL-r protein expression in olfactory bulb. HT modification of both apoE and LRP could have wide-spread effects on cellular function given LRP's manifold signaling functions.

Triglyceride-rich lipoproteins prime aortic endothelium for an enhanced inflammatory response to tumor necrosis factor-alpha.

High levels of triglyceride-rich lipoproteins (TGRLs) in blood are linked to development of atherosclerosis, yet the mechanisms by which these particles initiate inflammation of endothelium are unknown. TGRL isolated from human plasma during the postprandial state was examined for its capacity to bind to cultured human aortic endothelial cells (HAECs) and alter the acute inflammatory response to tumor necrosis factor-alpha. HAECs were repetitively incubated with dietary levels of freshly isolated TGRL for 2 hours per day for 1 to 3 days to mimic postprandial lipidemia. TGRL induced membrane upregulation of the low-density lipoprotein family receptors LRP and LR11, which was inhibited by the low-density lipoprotein receptor-associated protein-1. TGRLs alone did not elicit inflammation in HAECs but enhanced the inflammatory response via a 10-fold increase in sensitivity to cytokine stimulation. This was reflected by increased mitogen-activated protein kinase activation, nuclear translocation of NF-kappaB, amplified expression of endothelial selectin and VCAM-1, and a subsequent increase in monocyte-specific recruitment under shear flow as quantified in a microfabricated vascular mimetic device.

Estrogenic regulation of cholesterol biosynthesis and cell growth in DLD-1 human colon cancer cells.

OBJECTIVE: The main molecules of cholesterol homeostasis are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR), the key enzyme of the biosynthetic pathway, and the low-density lipoprotein receptor (LDL-R), which is responsible for the uptake of plasma lipoproteins. The increase in the endogenous cholesterol biosynthesis results in stimulation of DNA synthesis, while the inhibition of cholesterogenesis suppresses cell growth. Estrogens have been reported to regulate hepatic LDL-R expression and modulate cell proliferation in different tissues. In order to clarify the mechanisms of estrogenic growth control in colorectal carcinoma, we have investigated the effects of 17beta-estradiol exposure on LDL-R gene expression and its protein, as well as on HMG-CoAR gene expression, its protein as well as enzyme activity in the DLD-1 human colon cancer cell line. The effect of 17beta-estradiol on both cell growth and apoptosis in this cell line was also investigated. MATERIAL AND METHODS: LDL-R and HMG-CoAR gene expressions were determined using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in DLD-1 cells at different times and doses of 17beta-estradiol exposure. LDL-R and HMG-CoAR protein expression was detected by Western immunoblotting. HMG-CoAR activity was evaluated using a radiometric assay. Cell proliferation was measured by colorimetric MTT test and incorporation of [3H]-thymidine in DNA. Apoptotic death was estimated by DNA fragmentation analysis. RESULTS: Estrogens induced an early increase of LDL-R, at both mRNA and protein level, and later decreased HMG-CoAR activity and protein expression. DLD-1 cells treated with 17beta-estradiol exhibited inhibition of DNA synthesis and apoptosis. CONCLUSIONS: The results of this study suggest that estrogens regulate LDL-R and HMG-CoAR and influence cell growth and apoptosis in colon cancer cells.

Apolipoprotein E4 inhibits, and apolipoprotein E3 promotes neurite outgrowth in cultured adult mouse cortical neurons through the low-density lipoprotein receptor-related protein.

The apolipoprotein E4 (apoE4) genotype is a major risk factor for Alzheimer's disease (AD); however, the mechanism is unknown. We previously demonstrated that apoE isoforms differentially modulated neurite outgrowth in embryonic neurons and in neuronal cell lines. ApoE3 increased neurite outgrowth whereas apoE4 decreased outgrowth, suggesting that apoE4 may directly affect neurons in the brain. In the present study we examined the effects of apoE on neurite outgrowth from cultured adult mouse cortical neurons to examine if adult neurons respond the same way that embryonic cells do. The results from this study demonstrated that (1) cortical neurons derived from adult apoE-gene knockout (apoE KO) mice have significantly shorter neurites than neurons from adult wild-type (WT) mice; (2) incubation of cortical neurons from adult apoE KO mice with human apoE3 increased neurite outgrowth, whereas human apoE4 decreased outgrowth in a dose-dependent fashion; (3) the isoform specific effects were abolished by incubation of the neurons with either receptor associated protein (RAP) or lactoferrin, both of which block the interaction of apoE-containing lipoproteins with the low-density lipoprotein receptor-related protein (LRP). These data suggest a potential mechanism whereby apoE4 may play a role in regenerative failure and accelerate the development of AD.