Clusterin Research Articles

Clusterin exacerbates interleukin-1β-induced inflammation via suppressing PI3K/Akt pathway in human fibroblast-like synoviocytes of knee osteoarthritis

This study aimed to examine, a multifaceted chaperon-like protein exerting anti-inflammatory action, clusterin (CLU), mRNA and protein levels in the systemic and local joint environment of knee osteoarthritis (OA) patients and to determine whether CLU inhibited interleukin (IL)-1β-induced inflammation in knee OA fibroblast-like synoviocytes (FLSs) through modulating phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. CLU protein and mRNA expressions in the synovium and its protein levels in plasma and synovial fluid of knee OA patients were measured using immunohistochemistry, real-time PCR, and ELISA, respectively. Anti-inflammatory effect of CLU was further elucidated in knee OA FLSs treated with IL-1β in the absence or presence of CLU, CLU alone, or PI3K inhibitor (LY294002) along with IL-1β and CLU. In a clinical study, compared with knee OA patients without synovitis, CLU protein and mRNA were expressed in the synovium of knee OA patients with synovitis, especially those with high-grade, consistent with analyses of its plasma and synovial fluid levels. CLU mRNA and protein levels were both associated with synovitis severity. An in vitro study uncovered that CLU significantly alleviated IL-1β-induced overproduction of nitric oxide and IL-6 in knee OA FLSs. Furthermore, CLU significantly attenuated inflammation and extracellular matrix degradation induced by IL-1β via down-regulating expressions of IL-6, nuclear factor kappa B, and matrix metalloproteinase-13. Mechanistically, CLU significantly impeded IL-1β-induced Akt phosphorylation in knee OA FLSs, in line with addition of LY294002 along with IL-1β and CLU. These findings suggest that CLU may have potential as a novel therapeutic target for synovitis and cartilage destruction in knee OA.

Diagnostic performance of clusterin in hepatocellular carcinoma: A meta-analysis.

Clusterin (CLU) is a pleiotropic protein with numerous functions. It has recently attracted considerable attention owing to its association with cancer progression and metastasis. However, its role in hepatocellular carcinoma (HCC) has not been investigated. This meta-analysis is the first evaluation of the diagnostic performance of CLU in HCC. Articles published in PubMed, EMBASE, Web of Science, Wanfang Data Knowledge Service Platform, and China Science and Technology Journal Database until January 2022 were searched. Studies that reported the usefulness of CLU for the differentiation of HCC and non-HCC (e.g., liver cirrhosis, chronic hepatitis, and other benign liver disease) patients were selected. Alpha-fetoprotein (AFP) was used as a positive control in this study. The sensitivity, specificity, diagnostic odds ratio (DOR), and area under the curve (AUC) were compared between CLU and AFP. Eight articles including 811 participants were included. The pooled sensitivity (95% confidence interval (CI)), specificity (95% CI), DOR (95% CI), and AUC (95% CI) were: 0.86 (0.78-0.91), 0.85 (0.75-0.91), 35 (13-94), and 0.92 (0.89-0.94) for CLU; 0.74 (0.67-0.81), 0.89 (0.79-0.94), 22 (8-61), and 0.87 (0.84-0.90) for AFP; 0.93 (0.88-0.96), 0.85 (0.68-0.94), 75 (21-262), and 0.95 (0.92-0.96) for CLU + AFP, respectively. Compared with AFP, CLU showed higher sensitivity, DOR, and AUC, as well as similar specificity. The combination of CLU and AFP resulted in higher sensitivity, DOR, and AUC. Serum CLU is a better biomarker versus AFP for the diagnosis of HCC. The combination of CLU and AFP improved diagnostic performance.

Ginkgo biloba extract mitigates the neurotoxicity of AlCl3 in alzheimer rat’s model: role of apolipoprotein E4 and clusterin genes in stimulating ROS generation and apoptosis

Purpose Alzheimer’s disease (AD) appears as a result of an increase in the accumulation of amyloid beta peptide (Aβ) and a decrease in neurotransmitters (acetylcholine) within the brain cells which may be due to increase in acetylcholinesterase (AchE) activity and change in expression of Apolipoprotein E4 (ApoE4) and Clusterin (Clu) genes. The aim of the present study was using natural products such as Ginkgo biloba (G. biloba) extract that has the potential to reduce Aβ formation and increase AchE inhibition with its ability to save neuronal DNA from damage. Methods Sixty male aged rats were divided into six experimental groups exposed to AlCl3 to induce AD model and were treated with G. biloba extract. Collected brain tissues were used to assess the apoptosis rate, reactive oxygen species (ROS) generation, AchE inhibitory activity, expression alteration in ApoE4 and Clu genes, DNA fragmentations and gutathione peroxidase (GPx) activity.Results: The results exhibited that rats exposed to AlCl3 increased significantly rate of apoptosis, ROS formation, DNA fragmentation, up-regulation of ApoE4 and Clu genes as well as decrease of AchE inhibitory activity and GPx activity compared with those in control rats. However, treatment of AlCl3-rats with G. biloba extract improved the above neurotoxicity results induced by AlCl3 exposure. Conclusions It is therefore likely that G. biloba extract’s protective properties against AD are due to its ability to activate the response against oxidative stress.

The Most Promising Biomarkers of Allogeneic Kidney Transplant Rejection.

Clinical transplantology is a constantly evolving field of medicine. Kidney transplantation has become standard clinical practice, and it has a significant impact on reducing mortality and improving the quality of life of patients. Allogenic transplantation induces an immune response, which may lead to the rejection of the transplanted organ. The gold standard for evaluating rejection of the transplanted kidney by the recipient's organism is a biopsy of this organ. However, due to the high invasiveness of this procedure, alternative diagnostic methods are being sought. Therefore, the biomarkers may play an essential predictive role in transplant rejection. A review of the most promising biomarkers for early diagnosis and prognosis prediction of allogenic kidney transplant rejection summarizes novel data on neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), C-X-C motif chemokine 10 (CXCL-10), cystatin C (CysC), osteopontin (OPN), and clusterin (CLU) and analyses the dynamics of changes of the biomarkers mentioned above in kidney diseases and the mechanism of rejection of the transplanted kidney.

Evaluation of renal injury and function biomarkers, including symmetric dimethylarginine (SDMA), in the rat passive Heymann nephritis (PHN) model

Symmetric dimethylarginine (SDMA) is a serum biomarker of excretory renal function which consistently correlates with glomerular filtration rate (GFR) across multiple species including rats, dogs, and humans. In human and veterinary clinical settings SDMA demonstrates enhanced sensitivity for detection of declining renal function as compared to other serum biomarkers, but application in preclinical study designs thus far has been limited. The purpose of this study was to determine the performance of serum SDMA in a rat passive Heyman nephritis model of glomerulopathy. In addition to SDMA other biomarkers of excretory renal function were measured including serum creatinine (sCr), blood urea nitrogen (BUN), and cystatin C along with creatinine clearance. Urinary renal biomarkers including microalbumin (μALB), clusterin (CLU), cystatin C, kidney injury marker-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and osteopontin (OPN) were also measured. PHN was induced using commercial sheep anti-Fx1A serum. Tissue, serum, and urine were collected from groups of control and anti-Fx1A-treated animals for biomarker evaluation, hematology, urinalysis, serum biochemistry, and histologic examination of kidney. Over the course of a 28-day study, concentrations of the urinary biomarkers μALB, CLU, cystatin C, NGAL, KIM-1 and the serum biomarker cystatin C increased significantly in anti-Fx1A-treated rats as compared to controls but no significant increase in serum SDMA, sCr, BUN, or creatinine clearance were noted in anti-Fx1A-treated rats. Given lack of direct GFR measurement or significant change in the renal function biomarkers sCr, BUN, and creatinine clearance, it is unclear if GFR differed significantly between control and anti-Fx1A-treated rats in this study, though urinary biomarkers and histopathologic findings supported renal injury in anti-Fx1A-treated rats over the time course investigated. This study is among the first to investigate serum SDMA in a rat model relevant to preclinical safety assessment and serves to inform future experimental designs and biomarker selection when evaluation of glomerular injury is of priority.

Cytoplasmic Clusterin Suppresses Lung Cancer Metastasis by Inhibiting the ROCK1-ERK Axis.

Simple SummaryWe show that CLU, especially cytoplasmic precursor CLU, is downregulated in lung cancer and correlates with poor survival. The silencing of CLU promotes lung cancer cell migration and invasion, while the overexpression of CLU potently inhibits these phenomena. Interestingly, secretory CLU proteins are slightly decreased in lung cancer tissue and fail to exert similar anti-metastatic effects like cytoplasmic precursor CLU, demonstrating that cytoplasmic precursor CLU is the primary functional isoform of CLU, which exerts the anti-metastatic effects of lung cancer. Mechanistically, cytoplasmic precursor CLU binds ROCK1 to decrease phosphorylation of ERK1/2 by inhibiting the kinase activity of ROCK1, leading to an anti-metastatic effect in lung cancer cells. These findings reveal a novel insight into the function and regulation of cytoplasmic CLU in lung cancer, which might be a potential target for the diagnosis and treatment of metastatic lung cancer.Clusterin (CLU) is a heterodimeric glycoprotein that has been detected in diverse human tissues and implicated in many cellular processes. Accumulating evidence indicates that the expression of secreted CLU correlates with the progression of cancers. However, the molecular mechanisms underlying its tumor-suppressive roles are incompletely uncovered. In this study, we demonstrate that precursor CLU is widely downregulated in lung cancer tissue, in which secretory CLU proteins are slightly decreased. Impressively, overexpressing CLU potently inhibits the migration, invasion and metastasis of lung cancer cells, whereas silencing CLU promotes this behavior; however, it appears that secretory CLU fails to exert similar anti-metastatic effects. Interestingly, the cytoplasmic precursor CLU binds ROCK1 to abrogate the interaction between ROCK1 and ERK and impair ERK activity, leading to the suppression of lung cancer invasiveness. Meanwhile, the expression of CLU was remarkably diminished in lung cancer bone metastasis loci when compared with subcutaneous tumors in the mouse model and hardly detected in the bone metastasis loci of lung cancer patients when compared with the primary. These findings reveal a novel insight into the function and regulation of cytoplasmic CLU in lung cancer, which might be a potential target for the diagnosis and treatment of metastatic lung cancer.

The analysis of dynamic gene expression patterns in peripheral blood of multiple sclerosis patients indicates possible diagnostic and prognostic biomarkers

IntroductionAmong numerous invasive procedures for the research of biomarkers, blood-based indicators are regarded as marginally non-invasive procedures in the diagnosis and prognosis of demyelinating disorders, including multiple sclerosis (MS). In this study, we looked into the blood-derived gene expression profiles of patients with multiple sclerosis to investigate their clinical traits and linked them with dysregulated gene expressions to establish diagnostic and prognostic indicators. MethodsWe included 51 patients with relapsing-remitting MS (RRMS, n = 31), clinically isolated syndrome (CIS, n = 12), primary progressive MS (PPMS, n = 8) and a control group (n = 51). Using correlational analysis, the transcriptional patterns of chosen gene panels were examined and subsequently related with disease duration and the expanded disease disability score (EDSS). In addition, principal component analysis, univariate regression, and logistic regression analysis were employed to highlight distinct profiles of genes and prognosticate the excellent biomarkers of this illness. ResultsOur findings demonstrated that neurofilament light (NEFL), tumor necrosis factor α (TNF-α), Tau, and clusterin (CLU) were revealed to be increased in recruited patients, whereas the presenilin-1 (PSEN1) and cell-surface glycoprotein-44 (CD44) were downregulated. Principal Component Analysis revealed distinct patterns between the MS and control groups. Correlation analysis indicated co-dependent dysregulated genes and their differential expression with clinical findings. Furthermore, logistic regression demonstrated that Clusterin (AUC=0.940), NEFL (AUC=0.775), TNF-α (AUC=0.817), Tau (AUC=0.749), PSEN1 (AUC=0.6913), and CD44 (AUC=0.832) had diagnostic relevance. Following the univariate linear regression, a significant regression equation was found between EDSS and IGF-1 (R2 adj = 0.10844; p= 0.0060), APP (R2 adj = 0.1107; p= 0.0098), and PSEN1 (R2 adj = 0.1266; p=0.0102). ConclusionThis study exhibits dynamic gene expression patterns that represent the significance of specified genes that are prospective diagnostic and prognostic biomarkers for multiple sclerosis.

Targeted Analysis of Tears Revealed Specific Altered Metal Homeostasis in Age-Related Macular Degeneration.

PurposeSpecific altered metal homeostasis has been investigated in the tear film of age-related macular degeneration (AMD) patients considering that metal dyshomeostasis contributes to the production of free radicals, inflammation, and apoptosis and results in conformational changes of proteins.MethodsA multitargeted approach based on spectrophotometry and mass spectrometry techniques has been implemented to the multiplexed quantitation of lactoferrin (LF), S100 calcium binding protein A6 (S100A6), metallothionein 1A (MT1A), complement factor H (CFH), clusterin (CLU), amyloid precursor protein (APP), Mg, P, Na, Fe, Cu, Zn, and Ca, in the tear film from 60 subjects, 31 patients diagnosed with the dry form of AMD, and 29 healthy individualsResultsSignificant up-regulations of MT1A (1.9-fold) and S100A6 (1.4-fold) and down-regulations of LF (0.7-fold), Fe (0.6-fold), Mg (0.7-fold), and Cu (0.7-fold) were observed in AMD patients, when compared to control subjects. Of all the studied variables, only APP showed negative correlation with age in the AMD group. Also, positive correlations were observed for the variables Mg and Na, Cu and Mg, and P and Mg in both the AMD and control groups, whereas positive correlations were exclusively determined in the AMD group for Cu and LF, Na and Ca, and Mg and Ca. The panel constituted of MT1A, Na, and Mg predicts AMD disease in 73% of cases.ConclusionsThe different levels of target metals and (metallo-)proteins in the tear film suggest altered metal homeostasis in AMD patients. These observed pathophysiological changes may be related with the anomalous protein aggregation in the macula.

Molecular delineation, expression profiling, immune response, and anti-apoptotic function of a novel clusterin homolog from big-belly seahorse (Hippocampus abdominalis)

Clusterin (CLU) is a glycoprotein that contains α- and β-chains. CLU exerts multifunctional activities and plays a role in different cell signaling pathways that are associated with various diseases such as proteotoxic and oxidative stress, as well as cell death and survival. However, its role in marine teleost fish remains unclear. Therefore, the present study was carried out to characterize and investigate the immune responses and anti-apoptotic effects of CLU of the big-belly seahorse (Hippocampus abdominalis) (HaCLU) on oxidative stress-induced cell death. The HaCLU open reading frame was 1389 bp long and encoded a protein with 462 amino acids, a molecular weight of 51.28 kDa and an isoelectric point of 5.41. In-silico results demonstrated that HaCLU has a signal peptide in the 1–29 amino acid region, while the α- and β-chains were in the 34–227 and 228–455 amino acid regions, respectively. Multiple sequence alignment clarified the low homology of the α-chain with other orthologs. The highest HaCLU mRNA expression level was observed in the liver, followed by the heart, spleen, and brain tissues of healthy big-belly seahorses. Further, HaCLU mRNA expression level was elevated in the liver in response to different stimuli, including lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae. HaCLU potentiates cell viability and weakens chromatin condensation in the nucleus of FHM cells following H2O2-induced oxidative stress and subsequent cell death. HaCLU overexpression resulted in a reduced Bax/Bcl-2 mRNA expression ratio. This study revealed the role of HaCLU in immune regulation against pathogenic infections and its anti-apoptotic effects on oxidative stress-induced cell death.

Inhibition of Clusterin Represses Proliferation by Inducing Cellular Senescence in Pancreatic Cancer.

The outcome of pancreatic ductal adenocarcinoma (PDAC) is unsatisfactory, and the identification of novel therapeutic targets is urgently needed. Clinical studies on the antisense oligonucleotide that targets clusterin (CLU) expression have been conducted and have shown efficacy in other cancers. We aimed to investigate the effects of CLU in PDAC and the underlying mechanisms with a view to the clinical application of existing drugs. We knocked down CLU in PDAC cells and evaluated changes in cell proliferation. To elucidate the mechanism responsible for these changes, we performed western blot analysis, cell cycle assay, and senescence-associated β-galactosidase (SA-β-gal) staining. To evaluate the clinical significance of CLU, immunohistochemistry was performed, and CLU expression was analyzed in specimens resected from PDAC patients not treated with preoperative chemotherapy. Knockdown of CLU significantly decreased cell proliferation and did not induce apoptosis, but did induce cellular senescence by increasing the percentage of G1-phase and SA-β-gal staining-positive cells. A marker of DNA damage such as γH2AX and factors related to cellular senescence, such as p21 and the senescence-associated secretory phenotype, were upregulated by knockdown of CLU. CLU expression in resected PDAC specimens was located in the cytoplasm of tumor cells and revealed significantly better recurrence-free survival and overall survival in the CLU-low group than in the CLU-high group. We identified that CLU inhibition leads to cellular senescence in PDAC. Our findings suggest that CLU is a novel therapeutic target that contributes to the prognosis of PDAC by inducing cellular senescence.

Potential participation of CTRP6, a complement regulator, in the pathology of age related macular degeneration.

To investigate the localized expression of C1q/tumor necrosis factor related protein (CTRP) 6 in human age-related macular degeneration (AMD) retinal tissues. 4 AMD and 3 non-AMD whole eyes of Caucasian donors were used. Eyecups were excised at Eye Bank CorneaGen, Inc. To elucidate the effects of CTRP6, C3b was measured by an enzyme-linked immunosorbent-like assay. CFB versus CTRP6 competitive binding assay was applied to clarify the inhibition by CTRP6 of C3bBb complex formation. The cornea, iris, lens, and vitreous were removed and the eyes were cut into a posterior eye-cup including the retina, choroid, and sclera. Six-µm-thick serial sections of frozen samples underwent hematoxylin-eosin (HE) staining and indirect immunohistochemical staining using primary antibodies, anti-CTRP6, -CTRP5, -CTRP10, -Complement factor H (CFH) and -Clusterin (CLU). Results The two in vitro studies confirmed that CTRP6 has an inhibitory effect on alternative pathways of complement (APC) function and that the molecular target of CTRP6 is the inhibition of the formation of C3bBb. Localized expression for CTRP6 and CFH was found in the drusen of the AMD eyes, both associated with APC inhibition, CLU associated with membrane-attack complex (MAC) inhibition, and CTRP5 associated with retinal degeneration. The localized expression of CTRP6 in the drusen of AMD eyes may open a new insight into the possible involvement of APC regulatory factors in the pathogenesis of AMD, together with the known CFH so far analyzed solely as an APC inhibitor.

Deregulated Clusterin as a Marker of Bone Fragility: New Insights into the Pathophysiology of Osteoporosis.

Clusterin (CLU) is a secreted heterodimeric glycoprotein expressed in all organism fluids as well as in the intracellular matrix that plays key roles in several pathological processes. Its recent involvement in muscle degeneration of osteoporotic patients led to investigation of the role of CLU in bone metabolism, given the biochemical and biomechanical crosstalk of the bone–muscle unit. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis of CLU expression was performed in both osteoblasts and Peripheral Blood Mononuclear Cells (PBMCs) from osteoporotic patients (OP) and healthy individuals (CTR). Furthermore, immunohistochemical analysis on femoral head tissues and enzyme-linked immunosorbent assay (ELISA) in plasma samples were performed to investigate CLU expression pattern. Finally, genotyping of CLU rs11136000 polymorphism has also been performed by qRT-PCR assays to explore a possible association with CLU expression levels. Data obtained showed a significantly increased expression level of secreted CLU isoform in PBMCs and osteoblasts from OP patients. Immunohistochemical analysis confirms the increased expression of CLU in OP patients, both in osteocytes and osteoblasts, while plasma analysis reveals a statistically significant decrease of CLU levels. Unfortunately, no functional association between CLU expression levels and the presence of CLU rs11136000 polymorphism in OP patients was found. These data suggest a potential role played by CLU as a potential biomarker for the diagnosis and prognosis of OP progression.

RNA aptamers specific for transmembrane p24 trafficking protein 6 and Clusterin for the targeted delivery of imaging reagents and RNA therapeutics to human \u03b2 cells

The ability to detect and target β cells in vivo can substantially refine how diabetes is studied and treated. However, the lack of specific probes still hampers a precise characterization of human β cell mass and the delivery of therapeutics in clinical settings. Here, we report the identification of two RNA aptamers that specifically and selectively recognize mouse and human β cells. The putative targets of the two aptamers are transmembrane p24 trafficking protein 6 (TMED6) and clusterin (CLUS). When given systemically in immune deficient mice, these aptamers recognize the human islet graft producing a fluorescent signal proportional to the number of human islets transplanted. These aptamers cross-react with endogenous mouse β cells and allow monitoring the rejection of mouse islet allografts. Finally, once conjugated to saRNA specific for X-linked inhibitor of apoptosis (XIAP), they can efficiently transfect non-dissociated human islets, prevent early graft loss, and improve the efficacy of human islet transplantation in immunodeficient in mice.

Link of BIN1, CLU, and IDE Gene Polymorphisms with the Susceptibility of Alzheimer's Disease: Evidence from a Meta-analysis.

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder. The association of BIN1, CLU, and IDE genetic polymorphisms with AD risk have been evaluated overtimes that produced conflicting outcomes. We performed this meta-analysis to investigate the contribution of BIN1 (rs744373 and rs7561528), CLU (rs11136000 and rs9331888), and IDE (rs1887922) polymorphisms to AD risk. From a systemic literature search up to July 15, 2021, we included 25 studies with rs744373, 16 studies with rs7561528, 37 studies with rs11136000, 16 studies with rs9331888, and 4 studies with rs1887922. To analyze the correlation, we constructed seven genetic models that used odds ratio and 95% confidence intervals. We used RevMan 5.4 for meta-analysis. Our study suggests that BIN1 rs744373 is associated with a significantly increased risk of AD in five genetic models (OR>1). Again, CLU rs11136000 showed reduced association in all genetic models (OR<1). CLU rs9331888 revealed an increased association in two models (OR>1). The IDE rs1887922 showed significantly increased risk in four models (OR>1). From subgroup analysis, a significantly increased risk of AD was observed in Caucasians and Asians for BIN1 rs744373. Again, BIN1 rs7561528 showed a significantly enhanced risk of AD only in Caucasians. CLU rs11136000 showed significantly reduced risk in Caucasians but rs9331888 showed increased risk in the same ethnicity. Our meta-analysis confirms the association of BIN1 rs744373, CLU rs9331888, and IDE rs1887922 polymorphisms with an increased risk of AD, especially in Caucasians. Again, CLU rs11136000 is associated with reduced AD risk in the overall population and Caucasians.

Serum exosomal proteomics analysis of lung adenocarcinoma to discover new tumor markers

BackgroundAmong the most aggressive and rapidly lethal types of lung cancer, lung adenocarcinoma is the most common type. Exosomes, as a hot area, play an influential role in cancer. By using proteomics analysis, we aimed to identify potential markers of lung adenocarcinoma in serum.MethodsIn our study, we used the ultracentrifugation method to isolate serum exosomes. The Liquid chromatography-mass spectrometry (LC–MS) and bioinformatics analysis were used to identify potential serum exosomal proteins with altered expression among patients with advanced lung adenocarcinoma, early lung adenocarcinoma, and healthy controls. A western blot (WB) was performed to confirm the above differential expression levels in a separate serum sample-isolated exosome, and immunohistochemistry (IHC) staining was conducted to detect expression levels of the above differential proteins of serum exosomes in lung adenocarcinoma tissues and adjacent tissues. Furthermore, we compared different expression models of the above differential proteins in serum and exosomes.ResultAccording to the ITGAM (Integrin alpha M chain) and CLU (Clusterin) were differentially expressed in serum exosomes among different groups as well as tumor tissues and adjacent tissues. ITGAM was significantly and specifically enriched in exosomes. As compared to serum, CLU did not appear to be significantly enriched in exosomes. ITGAM and CLU were identified as serum exosomal protein markers of lung adenocarcinoma.ConclusionsThis study can provide novel ideas and a research basis for targeting lung adenocarcinoma treatment as a preliminary study.

Biomarkers for primary open-angle glaucoma progression

Glaucoma is a heterogeneous group of progressive optic neurodegenerative. Although most patients with primary open angle glaucoma (POAG) are stable for many years, certain subgroups of POAG patients could progress over time even with treatment. This study is to identify aqueous humor (AH) biomarkers that may be associated with disease progression in POAG patients. Gene differential expression study of prospectively collected AH from patients with stable or progressive POAG. Metagenomic deep sequencing (MDS) was performed on the aqueous fluid of 20 patients with stable POAG and 20 patients with progressive POAG. Differential gene expression analysis was performed to identify host transcriptome signatures. A total of 21 transcripts were differentially expressed between groups. Differential transcripts identified by MDS. Twenty transcripts were up-regulated and 1 transcript was down-regulated in progressive POAG patients compared to stable patients. Of those, 11 transcripts were eye-related, and 5 transcripts were related to glaucomatous phenotypes (Fibronectin type III domain containing 3B (FNDC3B), Clusterin (CLU), Proprotein convertase subtilisin/kexin type 6 (PCSK6), Cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1), and Rho guanine nucleotide exchange factor 4 (ARHGEF4)). Biomarkers associated with POAG progression can be identified from aqueous fluid. Identification of the biomarkers may improve glaucoma surveillance for progressive POAG.

Clusterin protects against Cr(VI)-induced oxidative stress-associated hepatotoxicity by mediating the Akt-Keap1-Nrf2 signaling pathway.

Hexavalent chromium [Cr(VI)] is a serious environmental pollutant that threatens human life. Cr(VI) is widely used in industrial processes such as metallurgy, leather processing, and electroplating, which can enter the human body through the respiratory or digestive tracts, thus causing a number of human disease, including inflammation and cancer. Although it has been confirmed that oxidative stress is one of the primary mechanism of liver injury caused by Cr(VI) exposure, the related toxic target and effective intervention measures have not been found. Clusterin (CLU) is an acute phase response protein with cytoprotective and apoptosis-delaying effects, and its expression has been confirmed to increase significantly after exposure to Cr(VI). In this study, our data clearly indicates that Cr(VI) is capable of causing hepatocytes damage through the production of large amounts of reactive oxygen species (ROS), causing an increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT). In contrast, over expression of CLU was able to inhibit ROS production and alleviate Cr(VI)-induced liver injury. The specific mechanisms are that CLU acts on the protein kinase B (PKB/Akt)-Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor E2-related factor 2 (Nrf2) signaling pathway to release Nrf2 into the nucleus. This is to initiate the expression of a downstream protein, heme oxygenase 1 (HO-1), thereby attenuating the ubiquitination ability of Keap1 with Nrf2. We also demonstrated that CLU could affect oxidative stress through the Akt/Nrf2 pathway, which reduced the production of ROS induced by Cr(VI) and protected against Cr(VI)-induced oxidative stress-associated hepatotoxicity. This study demonstrates a mechanism of Cr(VI)-induced hepatotoxicity and indicates that CLU as an intervention target of oxidative stress can provide valuable experimental basis for the prevention and treatment of occupational diseases in Cr(VI)-exposed population. Under the state of Cr(VI)-induced oxidative stress, CLU though phosphorylation Akt, leading to Nrf2 dissociation from Keap1. Activated Nrf2 entered the nucleus and formed the next step, thus binding to the structure of the antioxidant response element ARE, which activated HO-1, resulting in the decrease in intracellular ROS.

The functions of clusterin in renal mesenchymal stromal cells: Promotion of cell growth and regulation of macrophage activation

Clusterin (CLU) increases resistance to renal ischemia-reperfusion injury and promotes renal tissue repair. However, the mechanisms underlying of the renal protection of CLU remain unknown. Mesenchymal stromal cells (MSCs) may contribute to kidney cell turnover and injury repair. This study investigated the in vitro functions of CLU in kidney mesenchymal stromal cells (KMSCs). KMSCs were grown in plastic culture plates. Cell surface markers, apoptosis and phagocytosis were determined by flow cytometry, and CLU protein by Western blot. There were no differences in the expression of MSC markers (positive: CD133, Sca-1, CD44, CD117 and NG2, and negative: CD34, CD45, CD163, CD41, CD276, CD138, CD79a, CD146 and CD140b) and in the trilineage differentiation to chondrocytes, adipocytes and osteocytes between wild type (WT) and CLU knockout (KO) KMSCs. CLU was expressed intracellularly and secreted by WT KMSCs, and it was up-regulated by hypoxia. CLU did not prevent hypoxia-induced cell apoptosis but promoted cell growth in KMSC cultures. Furthermore, incubation with CLU-containing culture medium from WT KMSCs increased CD206 expression and phagocytic capacity of macrophages. In conclusion, our data for the first time demonstrate the function of CLU in the promotion of KMSCs proliferation, and it may be required for KMSCs-regulated macrophage M2 polarization and phagocytic activity.

Clusterin as a serum biomarker candidate contributes to the lung fibroblasts activation in chronic obstructive pulmonary disease.

Background:Fibrosis in the peripheral airways contributes to airflow limitation in patients with chronic obstructive pulmonary disease (COPD). However, the key proteins involved in its development are still poorly understood. Thus, we aimed to identify the differentially expressed proteins (DEPs) between smoker patients with and without COPD and elucidate the molecular mechanisms involved by investigating the effects of the identified biomarker candidate on lung fibroblasts.Methods:The potential DEPs were identified by isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic analysis. The messenger RNA and protein levels of clusterin (CLU) in COPD patients and 12% cigarette smoke extract (CSE)-treated human bronchial epithelial cells were determined at the indicated time points. Furthermore, an in vitro COPD model was established via the administration of 8% CSE to normal human lung fibroblasts (NHLFs) at indicated time points. The effects of CSE treatment and CLU silencing on proliferation and activation of lung fibroblasts were analyzed.Results:A total of 144 DEPs were identified between COPD patients and normal smokers. The iTRAQ-based proteomics and bioinformatics analyses identified CLU as a serum biomarker candidate. We also discovered that CLU levels were significantly increased (P < 0.0001) in Global Initiative for Obstructive Lung Disease II, III, and IV patients and correlated (P < 0.0001) with forced expiratory volume in 1 s (R = −0.7705), residual volume (RV) (R = 0.6281), RV/total lung capacity (R = 0.5454), and computerized tomography emphysema (R = 0.7878). Similarly, CLU levels were significantly increased in CSE-treated cells at indicated time points (P < 0.0001). The CSE treatment significantly inhibited the proliferation, promoted the inflammatory response, differentiation of NHLFs, and collagen matrix deposition, and induced the apoptosis of NHLFs; however, these effects were partially reversed by CLU silencing.Conclusion:Our findings suggest that CLU may play significant roles during airway fibrosis in COPD by regulating lung fibroblast activation.

Isotopic N,N-dimethyl leucine tags for absolute quantification of clusterin and apolipoprotein E in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia and one of the leading causes of death in the United States. In the past decades, extensive efforts have been devoted to biomarker discovery for early diagnosis and treatment of AD. Herein, this study aims to quantify clusterin (CLU) and apolipoprotein E (APOE) in blood samples from AD patients and evaluate these two proteins as potential biomarkers in AD diagnosis. In-house synthesized 5-plex isotopic N,N-dimethyl leucine (iDiLeu) tags were used to label target peptide standards at different concentrations to construct standard curves. Our study revealed that the levels of CLU and APOE exhibited clear differences in male vs. female AD groups but not in male vs. female non-AD groups. In contrast, the levels of serum CLU and APOE did not show statistically significant differences in the AD groups and non-AD groups. Principal component analysis (PCA) with CLU and APOE showed some separation between the AD and non-AD participants. Significance: Dissecting CLU and APOE heterogeneity in AD pathogenesis may therefore facilitate delineating the pathological relevance for sex-related pathways, leading to personalized medicine in the future. Collectively, this study introduces a cost-effective absolute quantitative proteomics strategy for target protein quantitation and lays the foundation for future investigation of CLU and APOE as potential biomarkers for AD. Significance statementAs blood-based biomarkers for AD diagnosis are cost-effective and introduce less invasiveness, discovery and validation of biomarkers in the blood samples of AD patients have become a hot topic in Alzheimer's and dementia research. Thus far, amyloid β (Aβ), total-tau and phosphorylated tau (p-tau) in blood show great accuracy and specificity in diagnosis of AD. However, the underlying mechanism of AD pathology remains to be elusive and complex. Besides these well studied proteins, many other proteins, such as clusterin (CLU) and apolipoprotein E (APOE) have also been found to be related to AD development. It has been implicated that these two proteins are involved in Aβ clearance and deposition. In this study, we measure the absolute concentrations of these two proteins in blood and shed some light on the potential roles of CLU and APOE in AD pathology. Dissecting CLU and APOE heterogeneity in AD pathogenesis may therefore facilitate delineating the pathological relevance for specific pathways between different genders, leading to personalized medicine in the future. Collectively, this study introduces a cost-effective absolute quantitative proteomics strategy for target protein quantitation and lays the foundation for future investigation of CLU and APOE as potential biomarkers for AD.