Low-density Lipoprotein Receptor-related Protein mRNA Research Articles

The effect of chronic stress and its preconditioning on spatial memory as well as hippocampal LRP1 and RAGE expression in a streptozotocin-induced rat model of Alzheimer’s disease

According to available evidence, prolonged or chronic exposure to stress is detrimental to various brain structures, including the hippocampus. The current study examined the expression of two critical blood-brain barrier receptors required for amyloid-beta clearance to understand better the mechanism by which chronic stress impairs learning and memory in patients with Alzheimer's disease (AD). Rats were randomly assigned to one of two groups in this study: experiment 1 and experiment 2. Each main group was then divided into four subgroups. Rats were bilaterally injected with streptozotocin (STZ, 3mg/kg, twice) using the intracerebroventricular (ICV) technique to induce the Alzheimer's model. Additionally, they were subjected to foot shock (1mA, 1Hz) for 10s every 60s (1h/day) for ten consecutive days prior to and following STZ injection. The Morris Water Maze (MWM) test was used to assess spatial learning and memory. Real-time PCR was used to determine Low-density lipoprotein receptor-related protein-1 (LRP1) and receptor for advanced glycation end-products (RAGE) mRNA levels in the hippocampus. Moreover, the animals' body weights were determined as physiological parameters in all groups. The results indicated that 10-day chronic electric foot shock stress reduced body weight, impaired spatial learning and memory, decreased hippocampal LRP1 mRNA expression, and increased hippocampal RAGE mRNA expression in a rat AD model. It can be concluded that chronic stress in conjunction with AD alters the expression of LRP1 and RAGE in the hippocampus. The findings pave the way for scientists to develop novel treatment strategies for AD.

Decreased low-density lipoprotein receptor-related protein 1 expression in pro-inflammatory monocytes is associated with subclinical atherosclerosis.

Subclinical atherosclerosis (SCA) occurs in asymptomatic individuals. Blood peripheral monocytes are involved in the development of atherosclerosis. Circulating monocytes acquire pro-inflammatory profiles, and they are involved in the early stages of atherosclerosis development. Low-density lipoprotein Receptor-related Protein 1 (LRP1) is expressed in monocytes, mainly in classical and intermediate subsets. Although LRP1 is highly expressed in macrophages and vascular smooth muscle cells (VSMCs) in atherosclerotic plaque formation, its expression in circulating monocytes has not been studied in SCA. The aim of this study was to characterize the LRP1 expression level in circulating monocytes of individuals with SCA and compared with individuals with low (LR) and intermediate (IR) risk of cardiovascular diseases, both without evidence of atherosclerotic lesions in carotid and coronary arteries. LRP1 and additional markers (CD11b, CD11c, and CD36) at cell surface of monocytes were analyzed by flow cytometry assays, whereas LRP1 and pro-inflammatory factors gene expressions were measured in isolated monocytes by quantitative RT-PCRs. Both LRP1 protein and LRP1 mRNA were significantly reduced in monocytes in SCA and IR respect to LR. Conversely, CD36, CD11b, and CD11c monocytic markers showed no significant changes between the different study groups. Finally, increased gene expressions of TNF-α and IL-1β were detected in monocytes of SCA, which were associated with decreased LRP1 expression at the cell surface in total monocytes. In summary, we propose that the decreased LRP1 expression at cell surface in total monocytes with pro-inflammatory profile is associated with the development of atherosclerosis in asymptomatic individuals.

Effect of moxibustion on expression of RAGE and LRP-1 and neuronal ultrastructure of frontal cortex and hippocampus in vascular dementia rats

To explore the effect of moxibustion of acupoints of Governor Vessel on learning-memory ability and expression of the receptor for advanced glycation end products (RAGE), low density lipoprotein (LDL) receptor related protein-1 (LRP-1), β-amyloid precursor protein (APP) and neuronal ultrastructure of the frontal cortex and hippocampus in vascular dementia (VD) rats, so as to investigate its underlying mechanisms in relieving VD. A total of 24 male SD rats were randomized into normal, model, and moxibustion groups (n＝8 rats in each group). The VD model was established by occlusion of the bilateral common carotid arteries for 20 min and reperfusion for 10 min which was repeated for 3 times. Moxibustion was applied to "Baihui" (GV20), "Dazhui"(GV14) and "Fengfu"(GV16)for 30 min, once daily for 4 weeks. The learning-memory ability was evaluated by using Morris water maze tests. At the end of experiments, the frontal lobe of cerebral cortex and hippocampus tissues were collected for detecting the expression of RAGE and LRP-1 proteins with Western blot, and RAGE, LRP-1 and APP mRNAs with quantitative real-time PCR, respectively. The ultrastructure of neurons in the frontal cortex and hippocampal CA1 region was observed with transmission electron microscopy. Following modeling, Morris water maze tests showed that the average escape latency of the model group and moxibustion group was significantly prolonged on the 4th day of modeling (P<0.01). The expression levels of RAGE protein and mRNA and APP mRNA were significantly increased (P<0.01), while those of LRP-1 protein and mRNA in the cortex and hippocampus remarkably decreased in the model group relevant to the normal group (P<0.01). Following moxibustion, modeling-induced increase of expression of RAGE protein and mRNA and APP mRNA, and down-regulation of expression of LRP-1 protein and mRNA were reversed in the moxibustion group relevant to the model group (P<0.01). Results of electron microscopy showed dilation of the mitochondria with disappearance of the cristae, partial vacuolar degeneration or dissolved external membrane, and cytoplasmic edema with basic disappearance of the rough endoplasmic reticulum and glycogen particles in neurons of the frontal cortex and hippocampal CA1 region after modeling, which was relatively milder in the moxibustion group. Moxibustion of acupoints of the Governor Vessel may reduce the content of APP in the frontal cortex and hippocampus by regulating the expression of RAGE and LRP-1 proteins and mRNA in VD rats.

Treadmill Exercise Ameliorates Spatial Learning and Memory Deficits Through Improving the Clearance of Peripheral and Central Amyloid-Beta Levels.

Aggregated amyloid beta (Aβ) peptides are believed to play a decisive role in the pathology of Alzheimer's disease (AD). Previous evidence suggested that exercise contributes to the improvement of cognitive decline and slows down pathogenesis of AD; however, the exact mechanisms for this have not been fully understood. Here, we evaluated the effect of a 4-week moderate treadmill exercise on spatial memory via central and peripheral Aβ clearance mechanisms following developed AD-like neuropathology induced by intra-hippocampal Aβ1-42 injection in male Wistar rats. We found Aβ1-42-treated animals showed spatial learning and memory impairment which was accompanied by increased levels of amyloid plaque load and soluble Aβ1-42 (sAβ1-42), decreased mRNA and protein expression of neprilysin (NEP), insulin degrading enzyme (IDE) and low-density lipoprotein receptor-related protein-1 (LRP-1) in the hippocampus. Aβ1-42-treated animals also exhibited a higher level of sAβ1-42 and a lower level of soluble LRP-1 (sLRP-1) in plasma, as well as a decreased level of LRP-1 mRNA and protein content in the liver. However, exercise training improved the spatial learning and memory deficits, reduced both plaque load and sAβ1-42 levels, and up-regulated expression of NEP, IDE, and LRP-1 in the hippocampus of Aβ1-42-treated animals. Aβ1-42-treated animals subjected to treadmill exercise also revealed decreased levels of sAβ1-42 and increased levels of sLRP-1 in plasma, as well as increased levels of LRP-1 mRNA and protein in the liver. In conclusion, our findings suggest that exercise-induced improvement in both of central and peripheral Aβ clearance are likely involved in ameliorating spatial learning and memory deficits in an animal model of AD. Future studies need to determine their relative contribution.

A common polymorphism decreases LRP1 mRNA stability and is associated with increased plasma factor VIII levels

The low-density lipoprotein receptor-related protein 1 (LRP1) gene is associated with increased levels of plasma factor VIII (FVIII). We aimed to explore eight functional genetic LRP1 variants for their potential roles in regulating FVIII levels and acute ischemic stroke (AIS). This genetic association study enrolled 192 patients with AIS and 134 controls. There were no significant differences in the genetic frequency of the eight functional single-nucleotide polymorphisms (SNPs) between the control and AIS groups. However, while analyzing the association between the eight SNPs and plasma FVIII levels, subjects with T/T genotype of rs1800137 (vs. CC+CT) were found to be associated with higher FVIII levels (23.5IU/dL; 95% confidence interval, 7.4–39.5IU/dL; P=0.0044) after adjusting for age, gender, estimated glomerular filtration rate, O blood type, inflammatory state, and body mass index.An analysis of the mRNA stability and abundance was designed and performed using minigene system transfected into HepG2 cells to assess the possible differences in mRNA stabilities between rs1800137 CC (rs1800137C) and TT (rs1800137T) genotypes. Site-directed mutagenesis revealed that rs1800137T accounts for the observed decrease in mRNA stability. The SNP rs1800137, located in exon 8, has been identified as an exon-splicing enhancer in silico. However, alternative splicing of LRP1 without inclusion of exon 8 was not identified. In transfected HepG2 cells, cycloheximide slowed down the degradation of the rs1800137T-containing minigene. These results demonstrate that synonymous SNP rs1800137 can lead to increased plasma FVIII levels due to decreased mRNA stability via translation-dependent mRNA degradation associated with codon optimality.

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 Concentration of Rosiglitazone Reduces Low‐density Lipoprotein Receptor‐Related Protein‐1 (LRP1) Levels Via Lysosomal Degradation in HepG2 Cells

LRP1 is a transmembrane receptor involved in the internalization of molecules by either transcellular transport or lysosomal degradation via clathrin. PPARγ is a nuclear receptor that transcribes several target genes involved in cell differentiation, lipid metabolism and glucose homeostasis. PPARγ activation has been shown to upregulate LRP1 mRNA and protein levels in HepG2 cells. However, overactivation of this nuclear receptor has exhibited a reduction in LRP1 protein levels. The aim of this research was to evaluate the effect of rosiglitazone (RGZ), a known PPARγ agonist, on LRP1 protein levels in HepG2 cells. Additionally, the LRP1 protein reduction mechanism in RGZ treated‐cells studying PPARγ activation, ubiquitin proteasome system and lysosomal degradation were evaluated.HepG2 cells were treated with RGZ (0.003 – 30 μM) to show no increase on LRP1 protein levels as reported in the literature. However, cells incubated for 24 and 48 hours with 30 μM of RGZ reduced LRP1 protein levels by 40 and 90%, respectively. PPARγ antagonism and ubiquitin proteasome system inhibition were studied in RGZ treated‐cells demonstrating no effect on LRP1 reduction. Bafilomycin A1, a lysosome degradation inhibitor, increased LRP1 protein levels up to 4‐fold in RGZ treated‐cells.These results suggest that overactivation of PPARγ reduces LRP1 protein levels via lysosome degradation. Further experiments are required to evaluate the mechanism involved in the effect of RGZ on LRP1 protein expression.

Clearance of matrix metalloproteinase-9 is dependent on low-density lipoprotein receptor-related protein-1 expression downregulated by microRNA-205 in human abdominal aortic aneurysm

Low-density lipoprotein receptor-related protein-1 (LRP1) has been suggested to be a crucial regulator in the pathogenesis of abdominal aortic aneurysm (AAA) from previous genome association and animal studies. Our prior study using human aortic samples has further revealed a significant reduction of LRP1 protein expression associated with AAA. However, the downregulation of LRP1 in the pathophysiology of AAA remained unresolved. We hypothesized that LRP1 downregulation may be mediated by microRNA (miR) and that LRP1 may function as a scavenger of matrix metalloproteinase-9 (MMP-9), a well-known protease for degradation of extracellular matrix proteins at the aortic wall for AAA pathogenesis. This study investigated the cause of LRP1 downregulation and its potential effect on AAA pathogenesis. An observational study of LRP1 protein, LRP1 messenger RNA (mRNA), and its three predicted miR candidates (miR-205, miR-338-5p, and miR-545-3p) was first performed in AAA compared with nonaneurysmal tissues from humans, followed by a functional study testing the effect on LRP1 expression of miR-205 overexpression and knockdown in human vascular smooth muscle cells (VSMCs) explant cultured from human abdominal aortic tissues. Lastly, another functional study was performed to test for the clearance of exogenous MMP-9 upon silencing of LRP1 in human VSMCs. From the observational study, significantly higher miR-205 (P< .001) and lower LRP1 protein (P< .001) expressions were found in human AAA tissues compared with nonaneurysmal aortic tissues, and no significant difference in LRP1 mRNA expression was observed. Further statistical analysis showed a significant negative correlation between miR-205 and LRP1 protein expressions (r=-0.65; P< .01). For the functional study, a significant downregulation of LRP1 protein expression was shown in miR-205-overexpressing VSMCs (P< .05), without any alteration in LRP1 mRNA expression. Moreover, a significantly reduced clearance of exogenous MMP-9 was observed in LRP1-silenced VSMCs (P< .05), and this difference in MMP-9 clearance was completely abolished with a pretreatment of anti-LRP1 antibody. Our study revealed the downregulation of LRP1 protein expression may be tightly regulated by miR-205 through translational inhibition in human VSMCs. Also, such LRP1 down-regulation in VSMCs may hinder the removal of pericellular MMP-9, leading to excess MMP-9 remaining in the extracellular matrix. Hence, the integrity of the vascular wall may be disrupted, promoting AAA formation.

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.

Selective Upregulation of Scavenger Receptors in and Around Demyelinating Areas in Multiple Sclerosis

Autoantibodies and complement opsonization have been implicated in the process of demyelination in the major human CNS demyelinating disease multiple sclerosis (MS), but scavenger receptors (SRs) may also play pathogenetic roles. We characterized SR mRNA and protein expression in postmortem brain tissue from 13 MS patients in relation to active demyelination. CD68, chemokine (C-X-C motif) ligand 16 (CXCL16), class A macrophage SR (SR-AI/II), LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1), FcγRIII, and LRP-1 (low-density lipoprotein receptor-related protein 1) mRNA were upregulated in the rims of chronic active MS lesions. CD68 and CXCL16 mRNA were also upregulated around chronic active MS lesions. By immunohistochemistry, CD68, CXCL16, and SR-AI/II were expressed by foamy macrophages in the rim and by ramified microglia around chronic active MS lesions. CXCL16 and SR-AI/II were also expressed by astrocytes in MS lesions and by primary human microglia and astrocytes in vitro. These data suggest that SRs are involved in myelin uptake in MS, and that upregulation of CD68, CXCL16, and SR-AI/II is one of the initial events in microglia as they initiate myelin phagocytosis. As demyelination continues, additional upregulation of LOX-1, FcγRIII, and LRP-1 may facilitate this process.

Low density lipoprotein receptor–related protein 1 is upregulated in epicardial fat from type 2 diabetes mellitus patients and correlates with glucose and triglyceride plasma levels

Lipoprotein receptor expression plays a crucial role in the pathophysiology of adipose tissue in in vivo models of diabetes. However, there are no studies in diabetic patients. The aims of this study were to analyze (a) low-density lipoprotein receptor-related protein 1 (LRP1) and very low-density lipoprotein receptor (VLDLR) expression in epicardial and subcutaneous fat from type 2 diabetes mellitus compared with nondiabetic patients and (b) the possible correlation between the expression of these receptors and plasmatic parameters. Adipose tissue biopsy samples were obtained from diabetic (n = 54) and nondiabetic patients (n = 22) undergoing cardiac surgery before the initiation of cardiopulmonary bypass. Adipose LRP1 and VLDLR expression was analyzed at mRNA level by real-time PCR and at protein level by Western blot analysis. Adipose samples were also subjected to lipid extraction, and fat cholesterol ester, triglyceride, and free cholesterol contents were analyzed by thin-layer chromatography. LRP1 expression was higher in epicardial fat from diabetic compared with nondiabetic patients (mRNA 17.63 ± 11.37 versus 7.01 ± 4.86; P = 0.02; protein 11.23 ± 7.23 versus 6.75 ± 5.02, P = 0.04). VLDLR expression was also higher in epicardial fat from diabetic patients but only at mRNA level (231.25 ± 207.57 versus 56.64 ± 45.64, P = 0.02). No differences were found in the expression of LRP1 or VLDLR in the subcutaneous fat from diabetic compared with nondiabetic patients. Epicardial LRP1 and VLDLR mRNA overexpression positively correlated with plasma triglyceride levels (R(2) = 0.50, P = 0.01 and R(2) = 0.44, P = 0.03, respectively) and epicardial LRP1 also correlated with plasma glucose levels (R(2) = 0.33, P = 0.03). These results suggest that epicardial overexpression of certain lipoprotein receptors such as LRP1 and VLDLR expression may play a key role in the alterations of lipid metabolism associated with type 2 diabetes mellitus.

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.

Association of ApoE and LRP mRNA levels with dementia and AD neuropathology

Inheritance of the ε4 allele of apolipoprotein E (ApoE) is the only confirmed and consistently replicated risk factor for late onset Alzheimer's disease (AD). ApoE is also a key ligand for low-density lipoprotein (LDL) receptor-related protein (LRP), a major neuronal low-density lipoprotein receptor. Despite the considerable converging evidence that implicates ApoE and LRP in the pathogenesis of AD, the precise mechanism by which ApoE and LRP modulate the risk for AD remains elusive. Moreover, studies investigating expression of ApoE and LRP in AD brain have reported variable and contradictory results. To overcome these inconsistencies, we studied the mRNA expression of ApoE and LRP in the postmortem brain of persons who died at different stages of dementia and AD-associated neuropathology relative to controls by quantitative polymerase chain reaction (qPCR) and Western blotting analyses. Clinical dementia rating scores were used as a measure of dementia severity, whereas, Braak neuropathological staging and neuritic plaque density were used as indexes of the neuropathological progression of AD. ApoE and LRP mRNA expression was significantly elevated in the postmortem inferior temporal gyrus (area 20) and the hippocampus from individuals with dementia compared with those with intact cognition. In addition to their strong association with the progression of cognitive dysfunction, LRP and ApoE mRNA levels were also positively correlated with increasing neuropathological hallmarks of AD. Additionally, Western blot analysis of ApoE protein expression in the hippocampus showed that the differential expression observed at the transcriptional level is also reflected at the protein level. Given the critical role played by LRP and ApoE in amyloid beta (Aβ) and cholesterol trafficking, increased expression of LRP and ApoE may not only disrupt cholesterol homeostasis but may also contribute to some of the neurobiological features of AD, including plaque deposition.

MicroRNA-205 inhibits tumor cell migration through down-regulating the expression of the LDL receptor-related protein 1

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor that plays critical roles in the pathogenesis of several human diseases including tumor metastasis and Alzheimer’s disease. However, mechanisms that regulate LRP1 expression under physiological and pathophysiological conditions are not unclear. In human cell lines, we found that miR-205 down-regulates the expression of LRP1 by targeting sequences in the 3′UTR of LRP1 mRNA. This effect was abolished by deleting the miR-205 seed site in the 3′UTR of LRP1. The ectopic expression of miR-205 also significantly mitigated migration of both U87 and SK-LU-1 cells. These results, for the first time, demonstrate that expression of human LRP1 is regulated in part by a specific miRNA, leading to decreased tumor cell migration.

Effects of ginkgo biloba extract EGb761 on expression of RAGE and LRP-1 in cerebral microvascular endothelial cells under chronic hypoxia and hypoglycemia

Alzheimer's disease (AD), characterized by accumulation of amyloid-beta protein (Abeta) in brain parenchyma, is closely associated with brain ischemia. Decreased clearance of Abeta from brain is the main cause of Abeta accumulation in sporadic AD. However, the mechanisms underlying ischemia-mediated AD pathogenesis remain unclear. The receptor for advanced end glycation products (RAGE) and low-density lipoprotein receptor-related protein-1 (LRP-1) expressed at blood brain barrier (BBB) are actively involved in Abeta clearance. RAGE is thought to be a primary transporter of Abeta across BBB into the brain from the systemic circulation, while LRP-1 mediates the transport of Abeta out of the brain. Ginkgo biloba extract EGb761, a traditional Chinese medicine, has been widely used in the treatment of AD. To investigate the effects of EGb761 on the expression of RAGE and LRP-1 in endothelial cells in response to ischemic injury, we cultured bEnd.3 cells, an immortalized mouse cerebral microvessel endothelial cell line, under a chronic hypoxic and hypoglycemic condition (CHH) to mimic ischemic injury of BBB, and then treated with EGb 761. We found that EGb 761 markedly ameliorated the damage (evaluated by MTT assay) from CHH. Moreover, we demonstrated that CHH led to a significant increase in the expression of RAGE both at the mRNA and protein levels at all times (24, 36, and 48 h), conversely; CHH induced a dramatic decrease in LRP-1 mRNA and protein expression at both 36 and 48 h. The results indicated that CHH has differential effects on the expression of RAGE and LRP-1. Furthermore, EGb761 significantly reversed CHH-induced upregulation of RAGE expression and downregulation of LRP-1 expression. Our findings suggest that EGb761 favor clearance of Abeta via regulating the expression of RAGE and LRP-1 during brain ischemia. This may provide a new insight into a possible molecular mechanism underlying brain ischemia-mediated AD pathogenesis, and potential therapeutic application of EGb 761 in treatment of AD.

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

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). Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed. 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. These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.

The Low-Density Lipoprotein Receptor-Related Protein Is a Pro-Survival Receptor in Schwann Cells: Possible Implications in Peripheral Nerve Injury

Schwann cells undergo phenotypic modulation in peripheral nerve injury. In the adult rodent, Schwann cells are resistant to death-promoting challenges. The responsible receptors and signaling pathways are incompletely understood. In this study, we demonstrate that low-density lipoprotein receptor-related protein-1 (LRP-1) is expressed in adult sciatic nerve. After crush injury, LRP-1 is lost from the axoplasm and substantially upregulated in Schwann cells. Increased LRP-1 mRNA expression was observed locally at the injury site in multiple forms of sciatic nerve injury, including crush injury, chronic constriction injury, and axotomy. Endogenously produced tumor necrosis factor-alpha (TNF-alpha) was mostly responsible for the increase in LRP-1 expression; this activity was reproduced by direct injection of TNF-alpha into injured nerves in the TNF-alpha gene knock-out mouse. TNF receptor II was primarily involved. TNF-alpha also increased LRP-1 mRNA in Schwann cells in primary culture. Silencing of Schwann cell LRP-1 with siRNA decreased phosphorylated Akt and increased activated caspase-3. Equivalent changes in cell signaling were observed in LRP-1-deficient murine embryonic fibroblasts. Schwann cell death was induced in vitro by serum withdrawal or TNF-alpha, to a greater extent when LRP-1 was silenced. Schwann cell death was induced in vivo by injecting the LRP-1 antagonist, receptor-associated protein, into axotomy sites in adult rats. These results support a model in which LRP-1 functions as a pro-survival receptor in Schwann cells.

Down-regulation of hepatic LDL receptor-related protein (LRP) in chronic renal failure

Chronic renal failure (CRF) is associated with premature atherosclerosis, impaired high-density lipoprotein (HDL)-mediated reverse cholesterol transport, depressed clearance, and elevated plasma concentrations of very low-density lipoprotein (VLDL), chylomicrons, and their atherogenic remnants. LDL receptor-related protein (LRP) is a member of the LDL receptor gene family that is heavily expressed in the liver, and mediates removal of at least 30 different ligands, including VLDL remnants (IDL) and chylomicron remnants. This study was conducted to test the hypothesis that the well-known defect in clearance of IDL and chylomicron remnants in CRF may be indicative of diminished hepatic LRP abundance. Hepatic tissue LRP mRNA abundance [reverse transcription-polymerase chain reaction (RT-PCR)] and protein abundance (Western blot analysis) were determined in rats 8 weeks after 5/6 nephrectomy (CRF group) or sham operation (control group). The CRF group exhibited hypertension, diminished creatinine clearance, increased plasma triglyceride concentration, and elevated total cholesterol-to-HDL cholesterol concentration ratio compared to the corresponding values found in the control group. This was associated with a significant down-regulation of hepatic LRP mRNA expression and immunodetectable LRP protein. CRF results in down-regulation of hepatic LRP. This abnormality can, at least in part, account for the previously documented elevation of plasma concentration and depressed clearance of chylomicron remnants and IDL in CRF.

Differential uptake of subfractions of triglyceride-rich lipoproteins by THP-1 macrophages

It is well known that raised plasma triglycerides (TG) are positively linked to the development of coronary heart disease. However, triglycerides circulate in a range of distinct lipoprotein subfractions and the relative atherogenicity of these subfractions is not clear. In this study, three fractions of triglyceride rich lipoprotein (TRL) were isolated from normolipidaemic males according to their differing Svedberg flotation ( S f) rates: chylomicron (CM, S f > 400), very low-density lipoprotein (VLDL)-1 ( S f 60–400) and VLDL-2 ( S f 20–60). These fractions were incubated with THP-1 monocyte-derived macrophages for determination of cholesterol and TG accumulation, in the presence and absence of the lipoprotein lipase (LPL) inhibitor orlistat. Expression of LDL receptor related protein (LRP) and apolipoprotein B48 receptor (apoB48R) was also examined in both differentiating monocytes, and monocyte-derived macrophages, incubated with TRL. VLDL-1 caused a significantly greater accumulation of TG within macrophages compared to VLDL-2. Binding studies also tended to show a greater preference for VLDL-1. No change in expression of LRP or apoB48R was observed in fully differentiated macrophages incubated with VLDL-1, VLDL-2 or CM, although a greater expression of LRP mRNA was observed in differentiating monocytes exposed to VLDL-1, compared to those incubated with CM or VLDL-2. TG loading in response to all three TRL fractions was blocked by orlistat, suggesting that it is likely that the major pathway for uptake of TG was hydrolysis by LPL. Calculations suggested that direct uptake of particles accounts for between 12 and 25% of total TAG uptake. In conclusion, THP monocyte-derived macrophages demonstrate a preference for VLDL-1, both through the LPL pathway and by direct uptake of whole particles.

Intracellular lipid accumulation, low-density lipoprotein receptor-related protein expression, and cell survival in vascular smooth muscle cells derived from normal and atherosclerotic human coronaries.

Vascular smooth muscle cell (VSMC) regulation during atherosclerotic plaque progression is determinant for plaque stability. To study lipid accumulation, low-density lipoprotein receptor-related protein (LRP) expression, and cell survival in VSMCs isolated from nonatherosclerotic areas (normal VSMCs) and advanced atherosclerotic plaques (plaque-VSMCs) of human coronaries. Normal or plaque-VSMCs were obtained from the intima by modification of the explant technique. Aggregated low-density lipoprotein (agLDL) (100 micro g mL(-1)) internalization induced higher intracellular cholesteryl ester (CE) accumulation in plaque-VSMC compared with normal VSMCs (89.28 +/- 6.1 vs. 60.34 +/- 4.1 micro g CE mg(-1) of protein; P < 0.05). This internalization was associated with LRP expression, as plaque-VSMCs show higher levels of LRP mRNA (6.06 +/- 0.55 vs. 3.87 +/- 0.28; P < 0.05) and LRP protein expression than normal VSMCs. However, plaque-VSMCs showed a lower proliferative response than normal VSMCs (6536 +/- 636 vs. 11151 +/- 815 c.p.m. [(3)H]thymidine; P < 0.05) and did not respond to platelet-derived growth factor BB (PDGF-BB) stimulus. In agreement, the Bcl(2)/BAX ratio was significantly lower in plaque-VSMCs compared with normal VSMCs (0.14 +/- 0.05 vs. 0.51 +/- 0.07; P < 0.05) and it was independent of lipid loading. These results indicate that higher intracellular lipid deposition in plaque-VSMCs is related to higher LRP expression levels. However, LRP-mediated agLDL internalization is not directly related to the reduced survival of plaque-VSMCs.