Receptor-related Protein Research Articles

LDL receptor related protein 2 to promote colorectal cancer metastasis via enhancing GSK3β/β-catenin signaling.

e15507 Background: LDL receptor related protein 2 (LRP2), a multi-ligand endocytic receptor, has been implicated in the tumourigenesis of various human cancers. However, the biological roles and underlying regulatory mechanism of LRP2 in CRC remain unclear. Methods: Real-time PCR (RT-PCR), western blot, and immunohistochemistry were used to examine LRP2 expression. Gene silencing and overexpressing efficiencies of LRP2 were confirmed by RT-PCR and western blot. Then wound-healing, migration assay and invasion assay were used to investigate the function of LRP2 in CRC cells. The protein expression and cellular localization of LRP2 and β-catenin were characterized by immunofluorescence staining. The nude mice tail vein metastasis model was established to observe the effect of LRP2 on the lung metastasis of CRC cells in vivo. In addition, gene set enrichment analysis was carried out to explore the potential mechanism of LRP2 in CRC. Results: LRP2 expression was highly upregulated in CRC compared with matched adjacent normal tissue. LRP2 overexpression was positively correlated with shorter overall survival in CRC patients. The biological function observation indicated that LRP2 promoted CRC metastasis in vitro and in vivo. Further molecular mechanism investigation demonstrated that LRP2 protein interacted with glycogen synthase kinase-3β (GSK-3β) and led to decreased expression of GSK3β at ser9, followed by enhanced β-catenin signaling pathway in CRC cells. Conclusions: Our study demonstrated that LRP2 could enhance the metastatic abilities of CRC cells in vitro and in vivo by inhibiting the expression of GSK3β and enhancing the activity of β-catenin signaling pathway, providing that LRP2 might be a novel therapeutic target for metastasis in CRC.

Optogenetic Control of the Canonical Wnt Signaling Pathway During Xenopus laevis Embryonic Development

Optogenetics uses light-inducible protein-protein interactions to precisely control the timing, localization, and intensity of signaling activity. The precise spatial and temporal resolution of this emerging technology has proven extremely attractive to the study of embryonic development, a program faithfully replicated to form the same organism from a single cell. We have previously performed a comparative study for optogenetic activation of receptor tyrosine kinases, where we found that the cytoplasm-to-membrane translocation-based optogenetic systems outperform the membrane-anchored dimerization systems in activating the receptor tyrosine kinase signaling in live Xenopus embryos. Here, we determine if this engineering strategy can be generalized to other signaling pathways involving membrane-bound receptors. As a proof of concept, we demonstrate that the cytoplasm-to-membrane translocation of the low-density lipoprotein receptor-related protein-6 (LRP6), a membrane-bound coreceptor for the canonical Wnt pathway, triggers Wnt activity. Optogenetic activation of LRP6 leads to axis duplication in developing Xenopus embryos, indicating that the cytoplasm-to-membrane translocation of the membrane-bound receptor could be a generalizable strategy for the construction of optogenetic systems.

Thymosin β4 protects against aortic aneurysm via endocytic regulation of growth factor signaling.

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic modulation which increases susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesized that Thymosin β4 (Tβ4) may function to maintain healthy vasculature throughout postnatal life. This was supported by the identification of an interaction with low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of platelet-derived growth factor BB (PDGF-BB) signaling and VSMC proliferation. LRP1 variants have been implicated by genome-wide association studies with risk of AAA and other arterial diseases. Tβ4-null mice displayed aortic VSMC and elastin defects that phenocopy those of LRP1 mutants, and their compromised vascular integrity predisposed them to Angiotensin II–induced aneurysm formation. Aneurysmal vessels were characterized by enhanced VSMC phenotypic modulation and augmented PDGFR-β signaling. In vitro, enhanced sensitivity to PDGF-BB upon loss of Tβ4 was associated with dysregulated endocytosis, with increased recycling and reduced lysosomal targeting of LRP1–PDGFR-β. Accordingly, the exacerbated aneurysmal phenotype in Tβ4-null mice was rescued upon treatment with the PDGFR-β antagonist Imatinib. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, and provides insights into the mechanisms of growth factor–controlled VSMC phenotypic modulation underlying aortic disease progression.

LRP1 is a master regulator of tau uptake and spread

The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer's disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE)1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation. 1 Goedert, M., Eisenberg, D. S. & Crowther, R. A. Propagation of Tau Aggregates and Neurodegeneration. Annu Rev Neurosci 40, 189-210, doi:10.1146/annurev-neuro-072116-031153 (2017). 2 Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82, 239-259, doi:10.1007/bf00308809 (1991). 3 de Calignon, A. et al. Propagation of tau pathology in a model of early Alzheimer's disease. Neuron 73, 685-697, doi:10.1016/j.neuron.2011.11.033 (2012). 4 Guo, J. L. et al. Unique pathological tau conformers from Alzheimer's brains transmit tau pathology in nontransgenic mice. J Exp Med 213, 2635-2654, doi:10.1084/jem.20160833 (2016).

Cpla2 and Lrp1 Deficiency Promote Severe Atherosclerosis Driven by LXRa/ABCA1 Dysfunction in Smooth Muscle Cells

Our group has identified smooth muscle cell targeted deficiency of LDL Receptor related protein 1 (LRP1), smLRP1-/-, resulting in accelerated aortic atherosclerosis. Activation of cytoplasmic phospholipase A2 (cPLA2) in these mice leaded to abolished LXRa/ABCA1 in vascular smooth muscle cells (VSMCs) and increased intracellular cholesterol accumulation. Based on our previous work we hypothesized that deficiency of cPLA2 would impede atherogenesis in the smLRP1-/- mouse model. Methods: Adult male smLRP1-/-;cPLA2-/-;LDLR-/- (Triple knockout) mice were placed on a high cholesterol diet (HCD) for 16 weeks and compared to age- and diet- matched sibling control smLRP1+/+;cPLA2-/-;LDLR-/- (Double KO) mice. Primary VSMC were explanted for in vitro migration, cell viability and proliferation studies when stimulated w/PDGF-BB. Histologic analysis was performed using en face whole aorta Oil red O (ORO) staining, as well as cross-sectional analysis of the aortic root with ORO, Picro Sirius Red, Alizarin Red and immunofluorescence. Immunoblot protein analysis was performed using lysed whole aortas. Data is represented as mean±SEM. Statistical analysis was performed using one- and two-way ANOVA with Tukey's correction. Results: Triple KO VSMC have decreased viable and proliferating cells but increased VSMC migration both compared to unstimulated and double KO controls. En face ORO analysis revealed increased lipid accumulation in Triple Knockout mice as compared to controls (60+/-3% vs 13+/-2%, p<0.001) (Figure 1). Uniquely, Triple knockout mice develop extensive necrotic cores and thin fibrous caps in atherosclerotic lesions in the aortic root (Figure 1). We found increased LXRa aggregates in triple KO mice and paradoxically ABCA1 is increased compared to controls observed by immunoblot and immunofluorescence tissue analysis of VSMC atherosclerotic tissues. Conclusions: Deficiency of cPLA2 in the smLRP1-/- mouse model induces LXRa increase and aggregation leading to rescue of ABCA1, but unexpectedly increases VSMC dysfunction and lipid accumulation within the plaque and generates more vulnerable plaque. Future studies will underpin the exact molecular mechanism that guide severe disease development in our Triple knockout mice via regulation of ABCA1.

Potential Roles of the WNT Signaling Pathway in Amyotrophic Lateral Sclerosis.

The WNT signaling pathway plays an important role in the physiological and pathophysiological processes of the central nervous system and the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We reviewed the literature pertinent to WNT/β–catenin signaling in ALS from cellular studies, animal models, and human clinical trials. WNT, WNT receptors, and other components of the WNT signaling pathway are expressed in both ALS patients and transgenic mice, and are involved in the pathogenesis of ALS. Studies have shown that abnormal activation of the WNT/β–catenin signaling pathway is related to neuronal degeneration and glial cell proliferation. WNT/Ca2+ signaling is associated with the pro–inflammatory phenotype of microglia; data on the muscle skeletal receptor Tyr kinase receptor in superoxide dismutase–1–G93A mice indicate that gene therapy is necessary for successful treatment of ALS. The varying profiles of lipoprotein receptor–related protein 4 antibodies in different ethnic groups suggest that individual treatment and multifactorial personalized approaches may be necessary for effective ALS therapy. In conclusion, the WNT signaling pathway is important to the ALS disease process, making it a likely therapeutic target.

Ruscogenin attenuated tight junction injury and tumor migration in colorectal liver metastasis mice via regulating TRAP1.

BackgroundThe effect of ruscogenin on the colorectal cancer is not clear yet. The study was applied to elucidate the mechanism of ruscogenin on colorectal cancer via regulating tumor necrosis factor receptor related protein 1 (TRAP1).MethodsHCT-116 cells were inoculated under the spleen capsule to establish the colorectal liver metastasis model. The group was divided into control, inoculation model, low dose (5 mg/kg), mediate dose (10 mg/kg), and high dose ruscogenin (20 mg/kg). The body and liver weight of the animals and tumor nodules were recorded. Western blot analysis and immunofluorescence assay were applied to indicate the alternation of tight junction, migration, and proliferation proteins.ResultsFollowing the inoculated with tumor cells, the mice in the inoculation group suffered from liver volume and weight decrease, as well as the increase of liver tumor volume (TV) and weight (TW). The administration of ruscogenin could obviously decrease body weight and increase liver weight in a dose-dependent manner. Meanwhile, 5, 10, 20 mg/kg ruscogenin could reduce the acreage of tumor nodule on liver, while the high dose 20 mg/kg ruscogenin could minimize the growth of tumor nodule. The intervention of ruscogenin could relieve the decreased expression of claudin-5, occludin, and ZO-1. The administration of ruscogenin could relieve the aggravated tight junction injury by the overexpression of TRAP1, while 20 mg/kg ruscogenin could not alleviate the tight junction injury already defused by the TRAP1 antibody in the colorectal cancer mice.ConclusionsRuscogenin could attenuate the tight junction injury via suppressing TRAP1 in the colorectal cancer mice.

Proteinuria converts hepatic heparan sulfate to an effective proprotein convertase subtilisin kexin type 9 enzyme binding partner

Hepatic uptake of triglyceride-rich remnant lipoproteins is mediated by the low-density lipoprotein receptor, a low-density lipoprotein receptor related protein and the heparan sulfate proteoglycan, syndecan-1. Heparan sulfate proteoglycan also mediates low-density lipoprotein receptor degradation by a regulator of cholesterol homeostasis, proprotein convertase subtilisin kexin type 9 (PCSK9), thereby hampering triglyceride-rich remnant lipoproteins uptake. In this study, we investigated the effects of proteinuria on PCSK9, hepatic heparan sulfate proteoglycan and plasma triglyceride-rich remnant lipoproteins. Adriamycin-injected rats developed proteinuria, elevated triglycerides and total cholesterol (all significantly increased). Proteinuria associated with triglycerides and total cholesterol and serum PCSK9 (all significant associations) without loss of the low-density lipoprotein receptor as evidenced by immunofluorescence staining and western blotting. In proteinuric rats, PCSK9 accumulated in sinusoids, whereas in control rats PCSK9 was localized in the cytoplasm of hepatocytes. Molecular profiling revealed that the heparan sulfate side chains of heparan sulfate proteoglycan to be hypersulfated in proteinuric rats. Competition assays revealed sulfation to be a major determinant for PCSK9 binding. PCSK9 partly colocalized with hypersulfated heparan sulfate in proteinuric rats, but not in control rats. Hence, proteinuria induces hypersulfated hepatic heparan sulfate proteoglycans, increasing their affinity to PCSK9. This might impair hepatic triglyceride-rich remnant lipoproteins uptake, causing proteinuria-associated dyslipidemia. Thus, our study reveals PCSK9/heparan sulfate may be a novel target to control dyslipidemia.

Ckip-1 regulates C3H10T1/2 mesenchymal cell proliferation and osteogenic differentiation via Lrp5.

Casein kinase-2 interaction protein-1 (Ckip-1) is a negative regulator of bone formation. The identification of novel Ckip-1-related targets and their associated signaling pathways that regulate mesenchymal stem cell (MSC) osteogenic differentiation is required. The present study aimed to evaluate the effects of Ckip-1 knockdown on C3H10T1/2 MSC proliferation and osteogenic differentiation, and to explore the role of the canonical Wnt-signaling receptor Lrp5. Ckip-1-knockdown (shCkip-1), Ckip-1-overexpression (Ckip-1) and their corresponding control [shCtrl and empty vector (EV), respectively] cell groups were used in the present study. Immunofluorescence localization of Ckip-1 was observed. The expression of the key molecules of the canonical Wnt signaling pathway was examined in C3H10T1/2 cells following osteogenic induction. Moreover, the effects of Lrp5 knockdown in the presence or absence of Ckip-1 knockdown were examined on C3H10T1/2 cell proliferation and osteogenic differentiation. The results indicated an increase in cell proliferation and osteogenic differentiation in the shCkip-1 group compared with the shCtrl group. The expression levels of LDL receptor related protein 5 (Lrp5), lymphoid enhancer binding factor 1 (Lef1) and transcription factor 1 in C3H10T1/2 cells were significantly increased in shCkip-1 cells following 7-day osteoinduction compared with shCtrl cells. Moreover, the involvement of Lrp5 in shCkip-1-induced osteogenic differentiation of C3H10T1/2 cells was further verified. The results indicated that Ckip-1 reduced C3H10T1/2 MSC proliferation and osteogenic differentiation via the canonical Wnt-signaling receptor Lrp5, which is essential for the improvement of bone tissue engineering.

Theranostic Design of Angiopep-2 Conjugated Hyaluronic Acid Nanoparticles (Thera-ANG-cHANPs) for Dual Targeting and Boosted Imaging of Glioma Cells.

Simple SummaryGlioblastoma multiforme is the most aggressive malignant brain tumor with poor patient prognosis. The presence of the blood-brain barrier and the complex tumor microenvironment impair the efficient accumulation of drugs and contrast agents, causing late diagnosis, inefficient treatment and monitoring. Functionalized theranostic nanoparticles are a valuable tool to modulate biodistribution of active agents, promoting their active delivery and selective accumulation for an earlier diagnosis and effective treatment, and provide simultaneous therapy and imaging for improved evaluation of treatment efficacy. In this work, we developed angiopep-2 functionalized crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and irinotecan (Thera-ANG-cHANPs) that were shown to boost relaxometric properties of Gd-DTPA by the effect of Hydrodenticity, improve the uptake of nanoparticles by the exploitation of angiopep-2 improved transport properties, and accelerate the therapeutic effect of Irinotecan.Glioblastoma multiforme (GBM) has a mean survival of only 15 months. Tumour heterogeneity and blood-brain barrier (BBB) mainly hinder the transport of active agents, leading to late diagnosis, ineffective therapy and inaccurate follow-up. The use of hydrogel nanoparticles, particularly hyaluronic acid as naturally occurring polymer of the extracellular matrix (ECM), has great potential in improving the transport of drug molecules and, furthermore, in facilitatating the early diagnosis by the effect of hydrodenticity enabling the T1 boosting of Gadolinium chelates for MRI. Here, crosslinked hyaluronic acid nanoparticles encapsulating gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and the chemotherapeutic agent irinotecan (Thera-cHANPs) are proposed as theranostic nanovectors, with improved MRI capacities. Irinotecan was selected since currently repurposed as an alternative compound to the poorly effective temozolomide (TMZ), generally approved as the gold standard in GBM clinical care. Also, active crossing and targeting are achieved by theranostic cHANPs decorated with angiopep-2 (Thera-ANG-cHANPs), a dual-targeting peptide interacting with low density lipoprotein receptor related protein-1(LRP-1) receptors overexpressed by both endothelial cells of the BBB and glioma cells. Results showed preserving the hydrodenticity effect in the advanced formulation and internalization by the active peptide-mediated uptake of Thera-cHANPs in U87 and GS-102 cells. Moreover, Thera-ANG-cHANPs proved to reduce ironotecan time response, showing a significant cytotoxic effect in 24 h instead of 48 h.

Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p.

Rationale: Cardiac fibrosis is an important feature of cardiac remodeling and is a hallmark of heart failure. Recent studies indicate that elevated IgE plays a causal role in pathological cardiac remodeling. However, the underlying mechanism of how IgE promotes cardiac fibrosis has not been fully elucidated.Methods and Results: To explore the function of IgE in cardiac fibrosis, we stimulated mouse primary cardiac fibroblasts (CFs) with IgE and found that both IgE receptor (FcεR1) and fibrosis related proteins were increased after IgE stimulation. Specific deletion of FcεR1 in CFs alleviated angiotensin II (Ang II)-induced cardiac fibrosis in mice. To investigate the mechanisms underlying the IgE-mediated cardiac fibrosis, deep miRNA-seq was performed. Bioinformatics and signaling pathway analysis revealed that IgE upregulated Col1a1 and Col3a1 expression in CFs by repressing miR-486a-5p, with Smad1 participating downstream of miR-486a-5p in this process. Lentivirus-mediated overexpression of miR-486a-5p was found to alleviate Ang II-induced myocardial interstitial fibrosis in mice. Moreover, miR-486-5p serum levels were lower in patients with heart failure than in healthy controls, and were negatively correlated with NT-proBNP levels.Conclusions: Our study demonstrates that elevated IgE promotes pathological cardiac fibrosis by modulating miR-486a-5p and downstream factors, such as Smad1. These findings suggest new targets for pathological cardiac fibrosis intervention.

From the low-density lipoprotein receptor–related protein 1 to neuropathic pain: a potentially novel target

This review describes the roles of the low-density lipoprotein receptor-related protein 1 (LRP-1) in inflammatory pathways, nerve nerve degeneration and -regeneration and in neuropathic pain. Induction of LRP-1 is able to reduce the activation of the proinflammatory NFκB-mediated pathway and the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase and p38 signaling pathways, in turn decreasing the production of inflammatory mediators. Low-density lipoprotein receptor-related protein 1 activation also decreases reactive astrogliosis and polarizes microglial cells and macrophages from a proinflammatory phenotype (M1) to an anti-inflammatory phenotype (M2), attenuating the neuroinflammatory environment. Low-density lipoprotein receptor-related protein 1 can also modulate the permeability of the blood-brain barrier and the blood-nerve barrier, thus regulating the infiltration of systemic insults and cells into the central and the peripheral nervous system, respectively. Furthermore, LRP-1 is involved in the maturation of oligodendrocytes and in the activation, migration, and repair phenotype of Schwann cells, therefore suggesting a major role in restoring the myelin sheaths upon injury. Low-density lipoprotein receptor-related protein 1 activation can indirectly decrease neurodegeneration and neuropathic pain by attenuation of the inflammatory environment. Moreover, LRP-1 agonists can directly promote neural cell survival and neurite sprouting, decrease cell death, and attenuate pain and neurological disorders by the inhibition of MAPK c-Jun N-terminal kinase and p38-pathway and activation of MAPK extracellular signal-regulated kinase pathway. In addition, activation of LRP-1 resulted in better outcomes for neuropathies such as Alzheimer disease, nerve injury, or diabetic peripheral neuropathy, attenuating neuropathic pain and improving cognitive functions. To summarize, LRP-1 plays an important role in the development of different experimental diseases of the nervous system, and it is emerging as a very interesting therapeutic target.

Regulation of tau internalization, degradation, and seeding by LRP1 reveals multiple pathways for tau catabolism

In Alzheimer's disease (AD), pathological forms of tau are transferred from cell to cell and “seed” aggregation of cytoplasmic tau. Phosphorylation of tau plays a key role in neurodegenerative tauopathies. In addition, apolipoprotein E (apoE), a major component of lipoproteins in the brain, is a genetic risk determinant for AD. The identification of the apoE receptor, low-density lipoprotein receptor–related protein 1 (LRP1), as an endocytic receptor for tau raises several questions about the role of LRP1 in tauopathies: is internalized tau, like other LRP1 ligands, delivered to lysosomes for degradation, and does LRP1 internalize pathological tau leading to cytosolic seeding? We found that LRP1 rapidly internalizes 125I-labeled tau, which is then efficiently degraded in lysosomal compartments. Surface plasmon resonance experiments confirm high affinity binding of tau and the tau microtubule-binding domain to LRP1. Interestingly, phosphorylated forms of recombinant tau bind weakly to LRP1 and are less efficiently internalized by LRP1. LRP1-mediated uptake of tau is inhibited by apoE, with the apoE4 isoform being the most potent inhibitor, likely because of its higher affinity for LRP1. Employing post-translationally–modified tau derived from brain lysates of human AD brain tissue, we found that LRP1-expressing cells, but not LRP1-deficient cells, promote cytosolic tau seeding in a process enhanced by apoE. These studies identify LRP1 as an endocytic receptor that binds and processes monomeric forms of tau leading to its degradation and promotes seeding by pathological forms of tau. The balance of these processes may be fundamental to the spread of neuropathology across the brain in AD.

Comprehensive molecular profiling to predict clinical outcomes in pancreatic cancer.

Background:Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among common cancers. The genomic landscape of PDAC is defined by four mutational pathways: kirsten rat sarcoma virus (KRAS), cellular tumor antigen p53 (TP53), cyclin dependent kinase inhibitor 2A (CDKN2A), and SMAD family member 4 (SMAD4). However, there is a paucity of data on the molecular features associated with clinical outcomes after surgery or chemotherapy.Methods:We performed comprehensive molecular characterization of tumor specimens from 83 patients with PDAC who received surgery, using whole-exome sequencing and ribonucleic acid sequencing on tumor and matched normal tissues derived from patients. We also systematically performed integrative analysis, combining genomic, transcriptomic, and clinical features to identify biomarkers and possible therapeutic targets.Results:KRAS (75%), TP53 (67%), CDKN2A (12%), SMAD4 (20%), and ring finger protein 43 (RNF43) (13%) were identified as significantly mutated genes. The tumor-specific transcriptome was classified into two clusters (tumor S1 and tumor S2), which resembled the Moffitt tumor classification. Tumor S1 displayed two distinct subclusters (S1-1 and S1-2). The transcriptome of tumor S1-1 overlapped with the exocrine-like (Collisson)/ADEX (Bailey) subtype, while tumor S1-2 mostly consisted of the classical (Collisson)/progenitor (Bailey) subtype. In the analysis of combinatorial gene alterations, concomitant mutations of KRAS with low-density lipoprotein receptor related protein 1B (LRP1B) were associated with significantly worse disease-free survival after surgery (p = 0.034). One patient (1.2%) was an ultrahypermutant with microsatellite instability. We also identified high protein kinase C lota (PRKCI) expression as an overlapping, poor prognostic marker between our dataset and the TCGA dataset.Conclusion:We identified potential prognostic biomarkers and therapeutic targets of patients with PDAC. Understanding these molecular aberrations that determine patient outcomes after surgery and chemotherapy has the potential to improve the treatment outcomes of PDAC patients.

Effects of normobaric cyclic hypoxia exposure on mesenchymal stem-cell differentiation-pilot study on bone parameters in elderly.

BACKGROUNDMesenchymal stem cells (MSC) of bone marrow are the progenitor of osteoblasts and adipocytes. MSC tend to differentiate into adipocytes, instead of osteoblasts, with aging. This favors the loss of bone mass and development of osteoporosis. Hypoxia induces hypoxia inducible factor 1α gene encoding transcription factor, which regulates the expression of genes related to energy metabolism and angiogenesis. That allows a better adaptation to low O2 conditions. Sustained hypoxia has negative effects on bone metabolism, favoring bone resorption. Yet, surprisingly, cyclic hypoxia (CH), short times of hypoxia followed by long times in normoxia, can modulate MSC differentiation and improve bone health in aging.AIMTo evaluate the CH effect on MSC differentiation, and whether it improves bone mineral density in elderly.METHODSMSC cultures were induced to differentiate into osteoblasts or adipocytes, in CH (3% O2 for 1, 2 or 4 h, 4 d a week). Extracellular-matrix mineralization and lipid-droplet formation were studied in MSC induced to differentiate into osteoblast or adipocytes, respectively. In addition, gene expression of marker genes, for osteogenesis or adipogenesis, have been quantified by quantitative real time polymerase chain reaction. The in vivo studies with elderly (> 75 years old; n = 10) were carried out in a hypoxia chamber, simulating an altitude of 2500 m above sea level, or in normoxia, for 18 wk (36 CH sessions of 16 min each). Percentages of fat mass and bone mineral density from whole body, trunk and right proximal femur (femoral, femoral neck and trochanter) were assessed, using dual-energy X-ray absorptiometry.RESULTSCH (4 h of hypoxic exposure) inhibited extracellular matrix mineralization and lipid-droplet formation in MSC induced to differentiate into osteoblasts or adipocytes, respectively. However, both parameters were not significantly affected by the other shorter hypoxia times assessed. The longest periods of hypoxia downregulated the expression of genes related to extracellular matrix formation, in MSC induced to differentiate into osteoblasts. Interestingly, osteocalcin (associated to energy metabolism) was upregulated. Vascular endothelial growth factor an expression and low-density lipoprotein receptor related protein 5/6/dickkopf Wnt signaling pathway inhibitor 1 (associated to Wnt/β-catenin pathway activation) increased in osteoblasts. Yet, they decreased in adipocytes after CH treatments, mainly with the longest hypoxia times. However, the same CH treatments increased the osteoprotegerin/receptor activator for nuclear factor kappa B ligand ratio in both cell types. An increase in total bone mineral density was observed in elderly people exposed to CH, but not in specific regions. The percentage of fat did not vary between groups.CONCLUSIONCH may have positive effects on bone health in the elderly, due to its possible inhibitory effect on bone resorption, by increasing the osteoprotegerin / receptor activator for nuclear factor kappa B ligand ratio.

Genome‐wide association analysis of neurofibrillary tangle burden identifies novel risk loci in the adult changes of thought (ACT) and the religious orders study and memory and aging project (ROSMAP) autopsy cohorts

AbstractBackgroundNeurofibrillary tangle (NFT) deposition is a hallmark for neuropathological diagnosis of late‐onset Alzheimer’s disease (LOAD). We were interested in identifying genetic variants associated with NFT burden in brain tissue from participants in the Adult Changes in Thought (ACT) and the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) autopsy cohorts.MethodIn ACT, NFT (paired helical filament (PHF)‐tau) is measured using histelide in frontal, temporal and parietal cortex. In ROSMAP, NFT burden is quantified by microscopic examination of silver‐stained slides from hippocampus and midfrontal, midtemporal, inferior parietal, and entorhinal cortex. In each cohort, we used Confirmatory Factor Analysis (CFA) to derive composites for NFT burden. We ran null models adjusting for sex and age at death; residuals were phenotypes for genetic analyses. We ran genome‐wide association studies (GWAS) of NFT burden using 1000G imputed data (NCBI; build 137) from European ancestry participants using Q‐ROADTRIPS which uses an empirical correlation matrix to account for population substructure. We performed meta‐analysis using the adaptively‐weighted Fisher’s method. Due to modest sample size and use of p‐values alone in meta‐analysis, we used conservative criteria for candidate loci, with minor allele frequency (MAF)&gt; 0.05, meta‐analytic p‐value &lt; 5 × 10−8, and p‐value &lt; 0.2 in each cohort. We used generalized linear models for the top SNPs to investigate direction of effects.ResultMean death ages were 89 (ROSMAP, n = 1,225) and 87 (ACT; n = 388); and 66% (ROSMAP) and 53% (ACT) were female. A single factor CFA model fit in ACT. A bi‐factor model with a secondary factor comprising midfrontal, midtemporal, and inferior parietal cortices was needed for ROSMAP. We identified two novel loci at LRP1B (Chr 2: rs10496895; Pmeta = 1.54 × 10−8) and upstream of ZHX2 (Chr 8: rs56258958; Pmeta = 4.9 × 10−8) apart from SNPs near APOE.ConclusionLDL receptor related protein 1B (LRP1B) has restricted expression in human tissues and binds to several extracellular ligands, including fibrinogen and APOE‐ carrying lipoproteins. Zinc‐finger and homeodomain protein 2 (ZHX2) is specifically expressed in neural progenitor cells during cortical neurogenesis. Further functional enrichment analysis is needed to determine whether these novel loci may provide targets for interventions to ameliorate LOAD.

In-silico identification of new inhibitors for Low-density lipoprotein receptor-related protein6 (LRP6)

Low-density lipoprotein receptor-related protein 6 (LRP6) is an important therapeutic target for diseases such as osteoporosis, Alzheimer, cancer, and neurodegenerative disease. Computational methods such as ligand-based and structure-based virtual screening have been introduced as an extremely efficient and accurate tool for finding new drug targets and candidates. In this study, we aimed to screen the National Cancer Institute (NCI) Diversity Set II and parts of the ZINC database by virtual screening to identify potential and safe compounds that can inhibit the LRP6 protein. By utilizing various screening methods such as rigid and flexible molecular docking and Lipinski’s rule of five, we identified 10 potential compounds. Then, their validity was further tested by molecular dynamics simulation and MMPBSA binding free energy calculations. Eventually, it was concluded that ZINC03954520, ZINC01729523, ZINC03898665, ZINC13152226, ZINC26730911 and ZINC01069082 compounds can be potential drug compounds for inhibiting LRP6 protein. These compounds in complex with β-propeller domains of LRP6 showed that they are capable of altering the backbone of these domains and interfere with their structural dynamics which may lead to the inhibition of the signal transmission. Communicated by Ramaswamy H. Sarma

Lrp6 Dynamic Expression in Tooth Development and Mutations in Oligodontia

Genes associated with the WNT pathway play an important role in the etiology of tooth agenesis. Low-density lipoprotein receptor–related protein 6 encoding gene (LRP6) is a recently defined gene that is associated with autosomal dominant inherited tooth agenesis. Here, we aimed to identify novel LRP6 mutations in patients with tooth agenesis and investigate the significance of Lrp6 during tooth development. Using whole-exome sequencing, we identified 4 novel LRP6 heterozygous mutations (c.2292G>A, c.195dup, c.1095dup, and c.1681C>T) in 4 of 77 oligodontia patients. Notably, a patient who carried a nonsense LRP6 mutation (c.2292G>A; p.W764*) presented a hypohidrotic ectodermal dysplasia phenotype. Preliminary functional studies, including bioinformatics analysis and TOP-/FOP-flash reporter assays, demonstrated that the activation of WNT/β-catenin signaling was compromised as a consequence of LRP6 mutations. RNAscope in situ hybridization revealed dynamic and special changes of Lrp6 expression during murine tooth development from E11.5 to E16.5. It was noteworthy that Lrp6 was specifically expressed in the epithelium at E11.5 to E13.5 but was expressed in both dental epithelium and dental papilla from E14.5 and persisted in both tissues at later stages. Our study broadens the mutation spectrum of human tooth agenesis and is the first to identify a LRP6 mutation in patients with hypohidrotic ectodermal dysplasia and reveal the dynamic expression pattern of Lrp6 during tooth development. Information from this study is conducive to understanding the functional significance of Lrp6 on the biological process of tooth development.

Characterization of Protein Unable to Bind Von Willebrand Factor in Recombinant Factor VIII Products: Can We Reduce Their Immunogenicity?

Introduction Therapeutic products with blood coagulation factor VIII (FVIII) have a wide range of protein contents per activity unit (or IU/mg, specific activity), available from previous studies (Lin et al, 2004; Butenas et al, 2009) and the prescribing information. The wide range of the specific activities seems to be more pronounced in recombinant FVIII products (rFVIII) compared to plasma-derived FVIII, implying presence of protein with altered structure and biochemical properties. In particular, rFVIII products were reported to contain a protein fraction (FVIII*), unable to bind von Willebrand factor (VWF) without functional activity (Lin et al, 2004;Ofosu et al 2012). Furthermore, FVIII* may represent a risk factor for development of FVIII inhibitors in Hemophilia A patients, as the binding to VWF was shown to reduce FVIII immunogenicity in a tissue culture and mice model systems (Gangadharan et al, 2017; Muczynski et al, 2018). Due to these reasons, FVIII* can be defined as an impurity, which needs to be understood and controlled in rFVIII products. Study objective To develop a methodology to isolate FVIII* from rFVIII samples and to characterize this fraction in various rFVIII products. Experimental design Using immobilized VWF affinity chromatography (IVAC) for analysis of FVIII samples, protein fractions collected from (i) the column flow-through (FVIIIFT, corresponding to FVIII*) and (ii) the column-bound and eluted fraction (FVIIIEL) were characterized using polyacrylamide gel electrophoresis (PAGE) gels followed by silver-staining and immunoblotting, FVIII activity test, surface plasmon resonance, mass spectrometry, and for plasma clearance in mice. Results A robust IVAC methodology was developed. Using this method, we isolated the FVIIIFT and FVIIIEL from all ten third-generation rFVIII products marketed in the USA by study time. These products represent all current pharmaceutical variants of rFVIII including full-size FVIII, B-domain deleted (truncated) FVIII, Fc-fused FVIII, single-chained FVIII, and PEGylated protein, including those with the extended plasma half-life. FVIIIFT was found in all rFVIII products at levels up to 22% of total protein. Compared to FVIIIEL, FVIIIFT had similar pattern of polypeptide bands by PAGE, but lower functional activity, significantly reduced sulfation at Tyr1680 important for VWF binding, moderately decreased interaction with recombinant cluster II of a low-density lipoprotein receptor related protein 1 (a major clearance receptor of FVIII), and approximately 3-times faster clearance in mice (Figure 1). Conclusions Our results show that the FVIII* structure is generally similar to that of the major fraction of rFVIII, while it differs by microheterogeneity in post-translational modifications and possibly local misfolding. The data suggest that upon administration of a rFVIII product in patients, its FVIII* fraction is rapidly removed from the circulation, resulting in a decrease of the effective dosage. Our findings demonstrate a potential of IVAC to control FVIII* fraction in rFVIII products, including its removal from the products during their manufacture. These applications may lead to achieving better quality and efficacy of rFVIII products including reduction of their immunogenicity for improving the care of Hemophilia A. Disclosures No relevant conflicts of interest to declare.

Serum anti-LRPAP1 is a common biomarker for digestive organ cancers and atherosclerotic diseases.

Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay‐linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti‐human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low‐density lipoprotein receptor–related protein–associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S‐transferase–fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis‐related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well‐known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking.