sLRP1 (Soluble Low-Density Lipoprotein Receptor-Related Protein 1): A Novel Biomarker for P2Y12 (P2Y Purinoceptor 12) Receptor Expression in Atherosclerotic Plaques.

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.

Efeitos do fragmento variável de cadeia única anti-LDL eletronegativa vetorizado em nanocápsulas na aterosclerose experimental / Effects of an anti-LDL(-) single chain fragment variable vectorized in nanocapsules in experimental atherosclerosis

As doenças cardiovasculares são a principal causa de mortalidade no mundo. A aterosclerose é a base fisiopatológica dessas doenças, sendo definida como um processo crônico-inflamatório multifatorial, resultando da interação de diferentes células como linfócitos, macrófagos, células endoteliais e células musculares lisas na parede arterial. A lipoproteína de baixa densidade eletronegativa [LDL(-)], uma subfração modificada da LDL nativa, desempenha um papel-chave na aterosclerose, uma vez que as modificações sofridas por esta partícula são capazes de induzir o acúmulo de ésteres de colesterol em macrófagos e a subsequente formação de células espumosas. O sistema imunológico é crucial no processo aterogênico e estratégias terapêuticas direcionadas à imunoregulação deste processo têm sido utilizadas como novas alternativas tanto na prevenção do desenvolvimento quanto da progressão desta doença. Dentre essas estratégias, destaca-se o uso de fragmentos de anticorpos como o scFv (do inglês, single chain fragment variable), que podem ainda estar conjugados a nanopartículas com o intuito de aumentar sua eficiência de ação no organismo. Diante do papel da LDL(-) na aterosclerose, este projeto objetivou avaliar os efeitos in vitro e in vivo de um sistema nanoestruturado contendo fragmentos scFv anti-LDL(-) derivatizados na superfície de nanocápsulas sobre macrófagos murinos e humanos primários e em camundongos knockout para o gene do receptor da LDL (Ldlr-/-) no desenvolvimento e na progressão dessa doença. Demonstrou-se que o tratamento de macrófagos com a formulação scFv anti-LDL(-)-MCMN-Zn diminuiu de forma significativa a captação de LDL(-), assim como a expressão de IL-1ß (mRNA e proteína) e MCP-1 (mRNA). Foi demonstrada a internalização da nanoformulação pelos macrófagos via diferentes mecanismos de endocitose, demonstrando seu potencial uso como carreador de fármacos. In vivo, a nanoformulação diminuiu de forma significativa a área da lesão aterosclerótica em camundongos Ldlr-/- submetidos à avaliação pela técnica de tomografia por emissão de pósitrons (do inglês, PET), utilizando o radiotraçador 18F-FDG (18F-desoxiglicose), associada à tomografia computadorizada (CT) com agente de contraste iodado, além da análise morfométrica das lesões no arco aórtico. O conjunto dos resultados obtidos evidenciou a ação ateroprotetora da formulação scFv anti-LDL(-)-MCMN-Zn, reforçando seu potencial como estratégia terapêutica na aterosclerose

Cardiovascular diseases are the leading cause of mortality worldwide. Atherosclerosis is the pathophysiological basis of these diseases, defined as a chronic inflammatory multifactorial process, resulting from the interaction of several cells such as lymphocytes macrophages, endothelial cells and smooth muscle cells within the arterial wall. The electronegative low-density lipoprotein [LDL(-)], a modified subfraction of native LDL, plays a key role in atherosclerosis, since its modifications are capable of inducing the accumulation of cholesteryl esters in macrophages and the subsequent foam cells formation. The immune system is crucial in atherogenic process and therapeutic strategies directed to the immunoregulation of this process have been used as a new alternative in the prevention of the development as well as the progression of this disease. Among these strategies, it is the use of antibody fragments such as scFv (single chain fragment variable), which may be also conjugated to nanoparticles in order to increase their efficiency in the body. Given the role of LDL(-) in atherosclerosis, the aim of this project was to evaluate the in vitro and in vivo effects of a nanostructured system containing scFv anti-LDL(-) fragments derivatized on the surface of nanocapsules on murine and human primary macrophages and in the development and progression of the disease in LDL receptor knockout mice (Ldlr-/-). It was demonstrated that the treatment of macrophages with scFv anti-LDL(-)-MCMN-Zn formulation significantly decreases the uptake of LDL(-) and the expression IL-1ß (mRNA and protein) and MCP-1 (mRNA). Moreover, the internalization of the nanoformulation by macrophages through different endocytosis mechanisms was shown, demonstrating its potential use as a nanocarrier. In vivo, the nanoformulation decreased the area of atherosclerotic lesions in Ldlr-/- mice evaluated by positron emission tomography with 18F-FDG associated with computed tomography with iodinated contrast agent (PET/CT), besides the lesion morphometric analysis at the aortic arch Thus, these data provide evidence of the atheroprotection action of the ateroprotection action of the scFv anti-LDL(-)-MCMN-Zn formulation, suggesting its promising use as a therapeutic strategy for atherosclerosis

GRP78 and alpha2-macroglobulin are new promising targets for metastatic castrate-resistant prostate cancer treatment

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Inverse relationship between raft LRP1 localization and non-raft ERK1,2/MMP9 activation in idiopathic dilated cardiomyopathy: potential impact in ventricular remodeling.

BACKGROUND: Idiopathic dilated cardiomyopathy (IDCM) is characterized by adverse ventricular remodeling attributed to altered activity of extracellular matrix metalloproteinase (MMP). MMP overactivation is linked to changes in extracellular signal-regulated kinases (ERK), reportedly modulated by the low-density lipoprotein receptor-related protein 1 (LRP1) receptor. The aim of this work was to compare the levels, membrane distribution and interactions of LRP1, ERK1,2 and MMP2/9 in control and IDCM myocardium. METHODS: Left ventricle samples from IDCM patients and control subjects were collected to analyze gene and protein expression by Real-time PCR and Western blot, respectively. Fractions enriched in cholesterol, Flotillin-1 and Caveolin-3 (rafts) were isolated from the remaining membrane (non-rafts) by sucrose gradient ultracentrifugation. We assessed the formation of LRP1-ERK1,2 complexes and MMP activity by immunoprecipitation and zymography, respectively. RESULTS: In control myocardium, LRP1 was exclusively found in non-rafts while activation of ERK1,2 was preferentially detected in rafts. LRP1/p-ERK1,2 complexes were almost undetectable in rafts and non-rafts. In contrast, in IDCM myocardium, LRP1 moved to rafts and ERK1,2 activation was found in raft and non-raft fractions. Moreover, LRP1/p-ERK1,2 complexes were also found in both membrane fractions, although the amount was higher in non-rafts where MMP9 overactivation was exclusively detected. CONCLUSIONS: The presented findings demonstrate a differential membrane compartmentalisation of ERK signaling in IDCM myocardium. The movement of LRP1 to rafts and the concomitant increase in non-raft-related ERK1,2/MMP9 activation may have crucial clinical implications in the progression of disease.

Characterization of the impairment of the uptake of apoptotic polymorphonuclear cells by monocyte subpopulations in systemic lupus erythematosus.

Efficient removal of apoptotic polymorphonuclear leukocytes (PMNs) is an important step in the resolution of inflammation, which protects tissues from the noxious contents of dying cells. While the impairment of apoptotic PMNs removal has been demonstrated for macrophages in systemic lupus erythematosus (SLE), recent studies show that monocytes are also capable of such phagocytosis, although their involvement in SLE is not clear. Therefore, we characterized phagocytosis of apoptotic PMNs by monocytes in 22 patients with SLE and 22 healthy controls. Using flow cytometry we demonstrate that in SLE peripheral blood monocytes show impaired phagocytosis of autologous apoptotic PMNs, while they efficiently engulf apoptotic PMNs isolated from healthy subjects. Monocytes CD14highCD16+ and CD14dimCD16+ more efficiently interacted with apoptotic neutrophils than CD16- cells both in SLE and healthy subjects. Monocytes in SLE showed modestly decreased expression of CD35 and CD91 and increased expression of T Cell Ig- and mucin-domain-containing molecule-3 (TIM-3); however, these differences were evident mainly in selected subsets of monocytes (CD16+) while defects in phagocytosis were observed in all monocyte subsets. Apoptotic cell-dependent induction of lipopolysaccharide (LPS) stimulated production of anti-inflammatory cytokine IL-10 by peripheral blood mononuclear cells (PBMC) was blunted in SLE while the production of pro-inflammatory cytokine TNF-α was unchanged.

A pilot study on low-density lipoprotein receptor-related protein-1 in Chinese patients with abdominal aortic aneurysm.

OBJECTIVE: There are no published data on the expression of low-density lipoprotein receptor-related protein 1 (LRP1) in human aortic tissues with abdominal aortic aneurysm (AAA), although some researchers have suggested that LRP1 may be a crucial regulator in the pathogenesis of AAA. The aim of this pilot study is to investigate LRP1 expression in aortic tissues from Chinese patients with AAA compared with normal control tissues. MATERIALS AND METHODS: This study used human abdominal aortic tissues with or without AAA as a research model. Aneurysmal abdominal aortas were collected from Chinese patients with AAA (n = 12) during open surgical aneurysmal repair at our institution, and normal control non-aneurysmal abdominal aortas were collected from Chinese healthy organ donors (n = 12) during organ transplantation. Protein expression of LRP1 was analyzed by western blotting and immunohistochemistry. RESULTS: LRP1 protein expression was significantly lower in AAA (mean LRP1AAA/LRP1(Normal Control) = 0.51 ± 0.28) than in normal control aortic tissues (mean LRP1(Normal Control)/LRP1(Normal Control) = 1 ± 0.18) in our small sample cohort (p < .001). No significant correlation was shown between LRP1 protein expression and the size of AAA (p > .05). CONCLUSIONS: Our pilot result suggests that a reduction in LRP1 protein expression may be associated with aneurysm progression.

The LDL receptor-related protein 1 (LRP1) regulates the PDGF signaling pathway by binding the protein phosphatase SHP-2 and modulating SHP-2- mediated PDGF signaling events.

BACKGROUND: The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor ß (PDGFRß) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRß pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRß signaling pathway by binding SHP-2 and competing with the PDGFRß for this molecule. METHODOLOGY/PRINCIPAL FINDINGS: To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRß kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRß. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that phosphorylated forms of LRP1 and PDGFRß compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.

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

BACKGROUND: Patients with Alzheimer's disease (AD) exhibit a failure in the clearance of amyloid ß peptides (Aß) 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. METHODS: 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. RESULT: 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. CONCLUSION: 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 Aß in brain tissues and thus exacerbate Alzheimer's-like pathology.

Surface markers of heterogeneous peripheral blood-derived smooth muscle progenitor cells.

OBJECTIVE: Smooth muscle progenitor cells (SMPCs) were intriguingly shown to act as a double-edged sword in the pathogenesis of atherosclerosis. To fully clarify the roles of SMPCs in atherosclerosis, a distinct panel of SMPC surface markers is mandatory to be developed. METHODS AND RESULTS: Microarray gene expression analyses were used to discover potential surface markers of SMPCs. In vitro and in vivo experiments documented that platelet-derived growth factor receptor-ß, carboxypeptidase M, carbonic anhydrase 12, receptor activity-modifying protein 1, and low-density lipoprotein receptor-related protein were the 5 specific surface markers regulating various SMPC functions, including migration, extracellular matrix formation, resistance to hypoxia, and anti-inflammation. In severe combined immunodeficiency/nonobese diabetic mice after femoral arterial wire injury, injected human peripheral blood mononuclear cells contributed to substantial amount of neointimal α-smooth muscle actin-positive cells, coexpressing platelet-derived growth factor receptor-ß, carboxypeptidase M, carbonic anhydrase 12, receptor activity-modifying protein 1, and low-density lipoprotein receptor-related protein. Based on these markers, a novel quantification assay was developed to enumerate circulating early SMPC. Early SMPC numbers were higher in patients with unstable angina compared with those with normal coronary angiograms. In patients with acute ST-elevation myocardial infarction, different patterns of serial early SMPC changes were noted, related to different clinical presentations. CONCLUSIONS: Surface markers of heterogeneous SMPCs exhibit various functions associated with atherosclerotic pathophysiology. Quantification of surface marker-defined SMPCs provides a platform for studying SMPCs in cardiovascular diseases.

Visualizing interaction of proteins relevant to Alzheimer's disease in intact cells.

To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer's disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid beta (Abeta) through sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Abeta production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein-protein-interaction in intact cells, which has proven to be a valuable method in AD research.

LRP1 controls biosynthetic and endocytic trafficking of neuronal prion protein.

The trafficking of normal cellular prion protein (PrPC) is believed to control its conversion to the altered conformation (designated PrPSc) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrPC on neurons is rapidly and constitutively endocytosed by means of coated pits, a property dependent upon basic amino acids at its N-terminus. Here, we show that low-density lipoprotein receptor-related protein 1 (LRP1), which binds to multiple ligands through basic motifs, associates with PrPC during its endocytosis and is functionally required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrPC in biosynthetic compartments, with a concomitant lowering of surface PrPC, suggesting that LRP1 expedites the trafficking of PrPC to the neuronal surface. PrPC and LRP1 can be co-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrPC binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 muM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface, and biosynthetic, trafficking of PrPC in neurons.

Lipoprotein receptor-related protein-1 mediates amyloid-beta-mediated cell death of cerebrovascular cells.

Inefficient clearance of A beta, caused by impaired blood-brain barrier crossing into the circulation, seems to be a major cause of A beta accumulation in the brain of late-onset Alzheimer's disease patients and hereditary cerebral hemorrhage with amyloidosis Dutch type. We observed association of receptor for advanced glycation end products, CD36, and low-density lipoprotein receptor (LDLR) with cerebral amyloid angiopathy in both Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis Dutch type brains and increased low-density lipoprotein receptor-related protein-1 (LRP-1) expression by perivascular cells in cerebral amyloid angiopathy. We investigated if these A beta receptors are involved in A beta internalization and in A beta-mediated cell death of human cerebrovascular cells and astrocytes. Expression of both the LRP-1 and LDLR by human brain pericytes and leptomeningeal smooth muscle cells, but not by astrocytes, increased on incubation with A beta. Receptor-associated protein specifically inhibited A beta-mediated up-regulation of LRP-1, but not of LDLR, and receptor-associated protein also decreased A beta internalization and A beta-mediated cell death. We conclude that especially LRP-1 and, to a minor extent, LDLR are involved in A beta internalization by and A beta-mediated cell death of cerebral perivascular cells. Although perivascular cells may adapt their A beta internalization capacity to the levels of A beta present, saturated LRP-1/LDLR-mediated uptake of A beta results in degeneration of perivascular cells.

Intrauterine growth restriction is associated with alterations in placental lipoprotein receptors and maternal lipoprotein composition.

Among other factors, fetal growth requires maternal supply of cholesterol. Cellular cholesterol uptake is mainly mediated by the LDL receptor (LDL-R) and the scavenger receptor family. We hypothesized that expression levels of key receptors of these families were regulated differently in placentas from IUGR pregnancies with varying degrees of severity. Third-trimester placentas from IUGR pregnancies with (IUGR-S) and without (IUGR-M) fetal hemodynamic changes and from control (AGA) pregnancies were studied. LDL-R, LDL-R-related protein (LRP-1), and scavenger receptor class B type I (SR-BI) mRNA and protein levels were measured. Cholesterol concentration and composition of lipoproteins were analyzed enzymatically and by lipid electrophoresis, respectively, in maternal and umbilical cord blood. LDL-R mRNA levels in IUGR-M were similar to AGA but lower (P < 0.05) in IUGR-S. In contrast, LDL-R protein was twofold (IUGR-M) and 1.8-fold (IUGR-S) higher (P < 0.05) than in the AGA group. LRP-1 mRNA and protein levels were not altered in the IUGR cases. SR-BI mRNA was unchanged in IUGR, but protein levels were lower (P < 0.05) in IUGR-S than in the other groups. Maternal plasma concentrations of LDL cholesterol were higher (P < 0.05) in the AGA group (188.5 +/- 23.6 mg/dl) than in the IUGR-S group (154.2 +/- 26.1). Electrophoretic mobility of the LDL fraction in maternal plasma demonstrated significant changes in migration toward higher values (AGA 0.95 +/- 0.06, IUGR-M 1.12 +/- 0.11, P < 0.001; IUGR-S 1.28 +/- 0.20, P = 0.002). We conclude that LDL-R and SR-BI levels are altered in IUGR pregnancies. These differences were associated with changes in LDL, but not HDL, mobility and cholesterol concentration in maternal circulation.

Human thyroid carcinoma cell invasion is controlled by the low density lipoprotein receptor-related protein-mediated clearance of urokinase plasminogen activator.

The low density lipoprotein receptor-related protein (LRP), a large scavenger receptor reported to mediate the uptake and degradation of various ligands, emerges as a promising receptor for targeting the invasive behaviour of human cancer cells. However, the accurate function of LRP during tumor invasion seems to be highly dependent on cellular context and remains controversial. The expression patterns of both this receptor and the main proteolytic systems involved in cell invasion were examined in two follicular thyroid carcinoma cell lines exhibiting different invasive phenotypes. We established that a low expression of LRP at the cell surface was associated to elevated extracellular MMP2 and urokinase plasminogen activator (uPA) activities as well as to high invasiveness properties. Surprisingly, neither exogenously added receptor-associated protein, an antagonist of LRP, nor LRP blocking antibodies significantly modified the amount of extracellular MMP2. Furthermore, the invasive phenotype of thyroid carcinoma cells was not related to their matrix metalloproteinases amount since different specific inhibitors of these proteases failed to affect the invasive properties of both cell lines. Additionally, blocking LRP-mediated clearance led to a further increase of the uPA amount and activities and to increased invasiveness in both cell lines. Finally thyroid carcinoma cells aggressiveness was widely increased by exogenous uPA; and anti-uPA antibodies treatments abolished both basal and receptor-associated protein-induced thyroid cell invasion. Overall our results identified the LRP-mediated clearance of uPA as one of the mechanisms involved during the control of human thyroid carcinoma cell invasion.

Soluble alpha2-macroglobulin receptor is increased in endotracheal aspirates from infants and children after cardiopulmonary bypass.

OBJECTIVE: Cytokine dysregulation contributes to the systemic inflammatory response after cardiopulmonary bypass. Clearance of cytokine binding proteins may be important in the resolution of inflammation. Our aim was to determine whether the cytokine binding protein alpha 2 -macroglobulin and its soluble receptor were upregulated in endotracheal aspirates from infants and children undergoing cardiopulmonary bypass. METHODS: Seventy tracheal aspirates were collected before and after cardiopulmonary bypass from 35 infants and children undergoing surgical correction of congenital heart defects. alpha 2 -Macroglobulin and the soluble alpha 2 -macroglobulin receptor were identified by Western blot. With the use of multi-analyte cytokine profiling, pro-inflammatory and anti-inflammatory cytokines were quantified, normalized to total protein, and expressed as ratios. Paired t tests and Wilcoxon signed-rank tests were performed between prebypass and postbypass samples. Correlations were examined among alpha 2 -macroglobulin, soluble alpha 2 -macroglobulin receptor, cytokine ratios, and the clinical variables of cardiopulmonary bypass, aortic crossclamp, and circulatory arrest times. RESULTS: alpha 2 -Macroglobulin increased by 50% (mean densitometry increase 82,683 +/- 184,594, P = .012), and soluble alpha 2 -macroglobulin receptor increased by 17% (mean densitometry increase 506,148 +/- 687,037, P = .0001) after cardiopulmonary bypass. The ratio of interleukin-8/interleukin-4 increased by 136% ( P = .0001), and interleukin-8/interleukin-10 increased by 102% ( P = .001). The increase in soluble alpha 2 -macroglobulin receptor was positively correlated with the ratios of interleukin-8/interleukin-4 and interleukin-8/interleukin-10. There were no statistically significant positive correlations between the increase in alpha 2 -macroglobulin or soluble alpha 2 -macroglobulin receptor and measured clinical variables. CONCLUSIONS: We report for the first time the upregulation of alpha 2 -macroglobulin and soluble alpha 2 -macroglobulin receptor in tracheal aspirates after cardiopulmonary bypass in infants and children. Soluble alpha 2 -macroglobulin receptor correlates with increased alpha 2 -macroglobulin and a disproportionate increase in pro-inflammatory to anti-inflammatory cytokine ratios.

Polimorfismo genético da apolipoproteína E na doença arterial periférica / Genetic polymorphism of apolipoprotein E in peripheral artery disease

Objetivo: este estudo teve corno objetivo analisar o polimorfismo genético da apolipoproteína E na doença arterial periférica obstrutiva ou aneurismática.Método: foram estudados 61 pacientes caucasóides do sexo masculino com sintomas clínicos e comprovação angiográfica de doença aterosclerótica, com idade entre 38 e 79 anos. O grupo-controle foi constituído por 59 indivíduos. Foram excluídos os indivíduos com doença renal, doença hepática ou diabetes melito. A análise dopolimorfismo genético da apolipoproteína E foi realizada por PCR(polimerase chain reaction) e RFLP (restriction fragment lenght polimorphism) com a endonuclease Hha I, identificando-se seis genótipos: 2/2, 2/3, 2/4, 3/3,3/4 e 4/4. Para análise estatística, foram aplicados teste exato de Fisher e odds ratio, com intervalo de confiança de 95 por cento, e admitiu-se erro a de até 5 por cento.Resultados: O alelo 3 mostrou-se mais freqüente em pacientes e controles, enquanto o alelo 4 apresentou maior prevalência nos controles em relação aos pacientes, embora sem diferença significante entre eles. Pacientes com doença arterial periférica obstrutiva mostrararo freqüência mais elevada do genótipo 3/4 em comparação àdoença arterial periférica aneurismática, embora sem diferença significante entre eles.Conclusão: o polimorfismo da apolipoproteína E-Hha I não foiassociada com a doença arterial periférica.

The low-density lipoprotein receptor-related protein associates with calnexin, calreticulin, and protein disulfide isomerase in receptor-associated-protein-deficient fibroblasts.

The low-density lipoprotein receptor-related protein (LRP) is a large (>600 kDa) multi-ligand-binding cell surface receptor that is now known to participate in a diverse range of cellular events. To accomplish this diverse role, LRP is composed of repetitive amino acid motifs consisting of complement-type and EGF precursor-type repeats. Within these repeats are six conserved cysteine residues that form the core disulfide bond structure of each repeat. To accommodate the intricate folding that such a complex structure dictates, a specialized chaperone is present in the endoplasmic reticulum (ER) called the receptor-associated protein (RAP) that binds to LRP immediately following its biosynthesis and assists in its exocytic transport. Interestingly, RAP -/- mice show reduced LRP expression in certain cell types, but not a more global affect on LRP expression that was expected. Such a tissue-restricted effect by RAP prompted an investigation if other ER chaperones associate with LRP to assist in its complex folding requirements and compensate for the absence of RAP in RAP -/- cells. Fibroblasts obtained from RAP -/- mice demonstrate similar LRP expression levels and subcellular distribution as RAP +/+ fibroblasts. Moreover, RAP -/- cells show an identical exocytic trafficking rate for LRP as RAP +/+ cells and comparable cell surface internalization kinetics. In RAP -/- cells, three well-known ER chaperones, calnexin, calreticulin, and protein disulfide isomerase (PDI), associate with LRP and likely compensate for the absence of RAP.

Factor VIII expression in liver disease.

Liver disease is associated with markedly elevated plasma factor VIII (FVIII) levels, whereas the synthesis of many other coagulation factors and proteins is reduced. In order to define the mechanism of FVIII increase, we have determined the expression levels of FVIII, both at mRNA and protein level, in patients with liver disease who underwent partial liver resection. In addition, the expression of von Willebrand factor (VWF) and low density lipoprotein receptor-related protein (LRP), proteins known for their ability to modulate FVIII plasma levels, were examined. Tissue samples for RNA extraction were obtained from 4 patients with cirrhosis, 9 patients with liver failure without cirrhosis and 6 patients with liver metastasis of a colon or rectum carcinoma (control group). In patients with liver cirrhosis hepatic FVIII and LRP mRNA levels were significantly lower than controls (p < or = 0.010), while VWF mRNA was significantly higher (p < or = 0.050). Immunohistochemical analysis revealed that cellular VWF protein distribution was also increased in cirrhotic livers compared to liver tissue from patients with non-cirrhotic liver disease. In cirrhotic tissue enlarged portal veins appeared to overgrow FVIII producing sinusoidal endothelial cells. Similarly, the number of LRP-producing cells appeared to be lower in cirrhotic tissue than in controls. The plasma concentration of both FVIII and VWF was significantly higher in patients with cirrhosis than control subjects (p = 0.038 and 0.010 respectively). These results demonstrate that elevated plasma FVIII levels in liver cirrhosis are associated with increased hepatic biosynthesis of VWF and decreased expression of LRP, rather than increased FVIII synthesis.

Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment.

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.