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Background: Progress in research on expression profiles in osteoarthritis (OA) has been limited to individual tissues within the joint, such as the synovium, cartilage, or meniscus. This study aimed to comprehensively analyze the common gene expression characteristics of various structures in OA and construct a diagnostic model. Methods: Three datasets were selected: synovium, meniscus, and knee joint cartilage. Modular clustering and differential analysis of genes were used for further functional analyses and the construction of protein networks. Signature genes with the highest diagnostic potential were identified and verified using external gene datasets. The expression of these genes was validated in clinical samples by Real-time (RT)-qPCR and immunohistochemistry (IHC) staining. This study investigated the status of immune cells in OA by examining their infiltration. Results: The merged OA dataset included 438 DEGs clustered into seven modules using WGCNA. The intersection of these DEGs with WGCNA modules identified 190 genes. Using Least Absolute Shrinkage and Selection Operator (LASSO) and Random Forest algorithms, nine signature genes were identified (CDADC1, PPFIBP1, ENO2, NOM1, SLC25A14, METTL2A, LINC01089, L3HYPDH, NPHP3), each demonstrating substantial diagnostic potential (areas under the curve from 0.701 to 0.925). Furthermore, dysregulation of various immune cells has also been observed. Conclusion: CDADC1, PPFIBP1, ENO2, NOM1, SLC25A14, METTL2A, LINC01089, L3HYPDH, NPHP3 demonstrated significant diagnostic efficacy in OA and are involved in immune cell infiltration.
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PURPOSE: There are currently no models for predicting hip fractures after stroke. This study wanted to investigate the risk factors leading to hip fracture in stroke patients and to establish a risk prediction model to visualize this risk. PATIENTS AND METHODS: We reviewed 439 stroke patients with or without hip fractures admitted to the Affiliated Hospital of Xuzhou Medical University from June 2014 to June 2017 as the training set, and collected 83 patients of the same type from the First Affiliated Hospital of Harbin Medical University and the Affiliated Hospital of Xuzhou Medical University from June 2020 to June 2023 as the testing set. Patients were divided into fracture group and non-fracture group based on the presence of hip fractures. Multivariate logistic regression analysis was used to screen for meaningful factors. Nomogram predicting the risk of hip fracture occurrence were created based on the multifactor analysis, and performance was evaluated using receiver operating characteristic curve (ROC), calibration curves, and decision curve analysis (DCA). A web calculator was created to facilitate a more convenient interactive experience for clinicians. RESULTS: In training set, there were 35 cases (7.9 %) of hip fractures after stroke, while in testing set, this data was 13 cases (15.6 %). In training set, univariate analysis showed significant differences between the two groups in the number of falls, smoking, hypertension, glucocorticoids, number of strokes, Mini-Mental State Examination (MMSE), visual acuity level, National Institute of Health stroke scale (NIHSS), Berg Balance Scale (BBS), and Stop Walking When Talking (SWWT) (P<0.05). Multivariate analysis showed that number of falls [OR=17.104, 95 % CI (3.727-78.489), P = 0.000], NIHSS [OR=1.565, 95 % CI (1.193-2.052), P = 0.001], SWWT [OR=12.080, 95 % CI (2.398-60.851), P = 0.003] were independent risk factors positively associated with new fractures. BMD [OR = 0.155, 95 % CI (0.044-0.546), P = 0.012] and BBS [OR = 0.840, 95 % CI (0.739-0.954), P = 0.007] were negatively associated with new fractures. The area under the curve (AUC) of nomogram were 0.939 (95 % CI: 0.748-0.943) and 0.980 (95 % CI: 0.886-1.000) in training and testing sets, respectively, and the calibration curves showed a high agreement between predicted and actual status with an area under the decision curve of 0.034 and 0.109, respectively. CONCLUSIONS: The number of falls, fracture history, low BBS score, high NIHSS score, and positive SWWT are risk factors for hip fracture after stroke. Based on this, a nomogram with high accuracy was developed and a web calculator (https://stroke.shinyapps.io/DynNomapp/) was created.
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OBJECTIVE: The continuously increasing extracellular matrix stiffness during intervertebral disc degeneration promotes disease progression. In an attempt to obtain novel treatment methods, this study aims to investigate the changes in nucleus pulposus cells under the stimulation of a stiff microenvironment. DESIGN: RNA sequencing and metabolomics experiments were combined to evaluate the primary nucleus pulposus and screen key targets under mechanical biological stimulation. Additionally, small molecules work in vitro were used to confirm the target regulatory effect and investigate the mechanism. In vivo, treatment effects were validated using a rat caudal vertebrae compression model. RESULTS: Our research results revealed that by activating TRPC6, hyperforin, a herbaceous extract can rescue the inflammatory phenotype caused by the stiff microenvironment, hence reducing intervertebral disc degeneration (IDD). Mechanically, it activates mitochondrial fission to inhibit PFKFB3. CONCLUSION: In summary, this study reveals the important bridging role of TRPC6 between mechanical stiffness, metabolism, and inflammation in the context of nucleus pulposus degeneration. TRPC6 activation with hyperforin may become a promising treatment for IDD.
Subject(s)
Extracellular Matrix , Intervertebral Disc Degeneration , Mitochondrial Dynamics , Nucleus Pulposus , Phloroglucinol , Rats, Sprague-Dawley , Animals , Nucleus Pulposus/metabolism , Nucleus Pulposus/pathology , Nucleus Pulposus/drug effects , Intervertebral Disc Degeneration/drug therapy , Intervertebral Disc Degeneration/pathology , Intervertebral Disc Degeneration/metabolism , Rats , Phloroglucinol/pharmacology , Phloroglucinol/analogs & derivatives , Phloroglucinol/therapeutic use , Mitochondrial Dynamics/drug effects , Extracellular Matrix/metabolism , Extracellular Matrix/drug effects , Male , Cells, Cultured , Humans , Terpenes/pharmacology , Terpenes/therapeutic use , TRPC Cation Channels/metabolism , Disease Models, Animal , Inflammation/drug therapyABSTRACT
Immune infiltration plays a crucial role in intervertebral disc degeneration (IDD). In this study, we explored the immune microenvironment of IDD through single-cell bioinformatics analysis. Three single-cell datasets were integrated into this study. Nucleus pulposus cells (NPCs) were divided into subgroups based on characteristic genes, and the role of each subgroup in the IDD process was analyzed through pseudo-time trajectory analysis. The hub genes were obtained using hdWGCNA, further identified by bulk datasets and pseudo-time sequence. The expression of the hub genes defined the NPCs related to immune infiltration, and the interaction between these NPCs and immunocytes was explored. The NPCs were divided into four subgroups: reserve NPCs, HCL-NPCs, response NPCs, and support NPCs, which, respectively, dominate the four processes of IDD: non, mild, moderate, and severe degeneration. SPP1 and ICAM1 were identified as the nucleus pulposus immune infiltration hub genes. Macrophages and myelocytes played pro-inflammatory roles in the SPP1-ICAM both-up NPC group through the SPP1-CD44 pathway and ICAM1-ITGB2 ligand-receptor pathway, respectively. At the same time, both-up NPCs sought self-help inflammation remission from neutrophils through the ANXA1-FPR1 pathway. The systematic analysis of the differentiation and immune infiltration landscapes helps to understand IDD's overall development process. Our data suggest that SPP1 and ICAM1 may be new targets for the treatment of inflammatory infiltration in IDD.
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Poly(ADP-ribose) polymerase (PARP) activation often indicates a disruptive signal to lipid metabolism, the physiological alteration of which may be implicated in the development of non-alcoholic fatty liver disease. The objective of this study was to evaluate the capability of [68Ga]DOTA-PARPi PET to detect hepatic PARP expression in a non-alcoholic steatohepatitis (NASH) mouse model. In this study, male C57BL/6 mice were subjected to a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for a 12-week period to establish preclinical NASH models. [68Ga]DOTA-PARPi PET imaging of the liver was conducted at the 12-week mark after CDAHFD feeding. Comprehensive histopathological analysis, covering hepatic steatosis, inflammation, fibrosis, along with blood biochemistry, was performed in both NASH models and control groups. Despite the induction of severe inflammation, steatosis and fibrosis in the liver of mice with the CDAHFD-NASH model, PET imaging of NASH with [68Ga]-DOTA-PARPi did not reveal a significantly higher uptake in NASH models compared to the control. This underscores the necessity for further development of new chelator-based PARP1 tracers with high binding affinity to enable the visualization of PARP1 changes in NASH pathology.
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BACKGROUND & AIMS: Persons with chronic HBV infection coinfected with HIV experience accelerated progression of liver fibrosis compared to those with HBV monoinfection. We aimed to determine whether HIV and its proteins promote HBV-induced liver fibrosis in HIV/HBV-coinfected cell culture models through HIF-1α and TGF-ß1 signaling. METHODS: The HBV-positive supernatant, purified HBV viral particles, HIV-positive supernatant, or HIV viral particles were directly incubated with cell lines or primary hepatocytes, hepatic stellate cells, and macrophages in mono or 3D spheroid coculture models. Cells were incubated with recombinant cytokines and HIV proteins including gp120. HBV sub-genomic constructs were transfected into NTCP-HepG2 cells. We also evaluated the effects of inhibitor of HIF-1α and HIV gp120 in a HBV carrier mouse model that was generated via hydrodynamic injection of the pAAV/HBV1.2 plasmid into the tail vein of wild-type C57BL/6 mice. RESULTS: We found that HIV and HIV gp120, through engagement with CCR5 and CXCR4 coreceptors, activate AKT and ERK signaling and subsequently upregulate hypoxia-inducible factor-1α (HIF-1α) to increase HBV-induced transforming growth factor-ß1 (TGF-ß1) and profibrogenic gene expression in hepatocytes and hepatic stellate cells. HIV gp120 exacerbates HBV X protein-mediated HIF-1α expression and liver fibrogenesis, which can be alleviated by inhibiting HIF-1α. Conversely, TGF-ß1 upregulates HIF-1α expression and HBV-induced liver fibrogenesis through the SMAD signaling pathway. HIF-1α small-interfering RNA transfection or the HIF-1α inhibitor (acriflavine) blocked HIV-, HBV-, and TGF-ß1-induced fibrogenesis. CONCLUSIONS: Our findings suggest that HIV coinfection exacerbates HBV-induced liver fibrogenesis through enhancement of the positive feedback between HIF-1α and TGF-ß1 via CCR5/CXCR4. HIF-1α represents a novel target for antifibrotic therapeutic development in HBV/HIV coinfection. IMPACT AND IMPLICATIONS: HIV coinfection accelerates the progression of liver fibrosis compared to HBV monoinfection, even among patients with successful suppression of viral load, and there is no sufficient treatment for this disease process. In this study, we found that HIV viral particles and specifically HIV gp120 promote HBV-induced hepatic fibrogenesis via enhancement of the positive feedback between HIF-1α and TGF-ß1, which can be ameliorated by inhibition of HIF-1α. These findings suggest that targeting the HIF-1α pathway can reduce liver fibrogenesis in patients with HIV and HBV coinfection.
Subject(s)
Coinfection , HIV Infections , Hepatitis B virus , Hypoxia-Inducible Factor 1, alpha Subunit , Liver Cirrhosis , Signal Transduction , Transforming Growth Factor beta1 , Animals , Transforming Growth Factor beta1/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Mice , Liver Cirrhosis/metabolism , Liver Cirrhosis/virology , Liver Cirrhosis/pathology , Humans , HIV Infections/complications , HIV Infections/metabolism , HIV Infections/pathology , Hepatitis B virus/genetics , Coinfection/virology , Mice, Inbred C57BL , Hepatitis B, Chronic/complications , Hepatitis B, Chronic/metabolism , Hepatitis B, Chronic/pathology , Hepatitis B, Chronic/virology , HIV Envelope Protein gp120/metabolism , Hepatocytes/metabolism , Hepatocytes/virology , Hepatocytes/pathology , Hepatic Stellate Cells/metabolism , Hepatic Stellate Cells/virology , Disease Models, Animal , Hep G2 Cells , MaleABSTRACT
Parkinson's disease (PD) is one of the most highly debilitating neurodegenerative disorders, which affects millions of people worldwide, and leucine-rich repeat kinase 2 (LRRK2) mutations have been involved in the pathogenesis of PD. Developing a potent LRRK2 positron emission tomography (PET) tracer would allow for in vivo visualization of LRRK2 distribution and expression in PD patients. In this work, we present the facile synthesis of two potent and selective LRRK2 radioligands [11C]3 ([11C]PF-06447475) and [18F]4 ([18F]PF-06455943). Both radioligands exhibited favorable brain uptake and specific bindings in rodent autoradiography and PET imaging studies. More importantly, [18F]4 demonstrated significantly higher brain uptake in the transgenic LRRK2-G2019S mutant and lipopolysaccharide (LPS)-injected mouse models. This work may serve as a roadmap for the future design of potent LRRK2 PET tracers.
Subject(s)
Morpholines , Nitriles , Parkinson Disease , Pyrimidines , Mice , Animals , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Leucine , Positron-Emission Tomography/methods , Parkinson Disease/metabolism , MutationABSTRACT
BACKGROUND: Percutaneous kyphoplasty (PKP) or percutaneous short-segment fixation (PSSF) is often used to treat Kummell's disease. However, it is not clear which treatment is better for patients. OBJECTIVE: To retrospectively compare the clinical efficacy of PVP and PSSF for the treatment of Kummell's disease. METHOD: 60 patients were involved in this research and the period of follow-up was at least 2 years. 27 of them were treated with PVP (Group I) and the rest who received PSSF (Group II). The visual analog scale (VAS) and radiographic indexes of each participant had been measured preoperatively as well as 1 week, 3 months, and 2 years postoperatively. Additionally, the Oswestry Disability Index (ODI) scores were assessed at the last time point. RESULTS: Comparing the two groups, no statistical significance was found among all parameters preoperatively. The time of operations and blood loss is less in Group I. At each time point after operation, the imaging indices in Group II are lower (P< 0.05). One week after treatments, the VAS scores are lower in Group I, and similarly, 3 months are the same (P< 0.05), while VAS are similar at the last time point. In the aspect of ODI scores, they are lower in Group II during long-term follow-up. CONCLUSION: For the treatment of Kummell's disease, both PVP and PSSF have been found to be effective. PVP can provide rapid pain relief with a shorter operation time. However, in cases with severe kyphosis deformity, PSSF should be given priority.
Subject(s)
Fractures, Compression , Kyphoplasty , Spinal Fractures , Spondylosis , Vertebroplasty , Humans , Follow-Up Studies , Vertebroplasty/adverse effects , Retrospective Studies , Spinal Fractures/surgery , Kyphoplasty/methods , Treatment Outcome , Fractures, Compression/surgeryABSTRACT
Fibroblast growth factor 21 (FGF21) is a glucose and lipid metabolic regulator. Recent research revealed that FGF21 was also induced by inflammatory stimuli. Its role in inflammatory bowel disease (IBD) has not been investigated. In this study, an experimental IBD model was established in FGF21 knockout (KO) and wild-type (WT) mice by adding 2.5% (wt/vol) dextran sodium sulfate (DSS) to their drinking water for 7 days. The severity of the colitis and the inflammation of the mouse colon tissues were analyzed. In WT mice, acute DSS treatment induced an elevation in plasma FGF21 and a significant loss of body weight in a time-dependent manner. Surprisingly, the loss of body weight and the severity of the colitis induced by DSS treatment in WT mice were significantly attenuated in FGF21 KO mice. Colon and circulating pro-inflammatory factors were significantly lower in the FGF21 KO mice compared to the WT mice. As shown by BrdU staining, the FGF21 KO mice demonstrated increased colonic epithelial cell proliferation. DSS treatment reduced intestinal Paneth cell and goblet cell numbers in the WT mice, and this effect was attenuated in the FGF21 KO mice. Mechanistically, FGF21 deficiency significantly increased the signal transducer and activator of transcription (STAT)-3 activation in intestinal epithelial cells and increased the expression of IL-22. Further study showed that the expression of suppressor of cytokine signaling-2/3 (SOCS 2/3), a known feedback inhibitor of STAT3, was significantly inhibited in the DSS-treated FGF2 KO mice compared to the WT mice. We conclude that FGF21 deficiency attenuated the severity of DSS-induced acute colitis, which is likely mediated by enhancing the activation of the IL-22-STAT3 signaling pathway in intestinal epithelial cells.
Subject(s)
Colitis , Inflammatory Bowel Diseases , Animals , Mice , Colitis/chemically induced , Body Weight , Interleukin-22ABSTRACT
BACKGROUND: The differences in host antiviral gene expression and disease severity between vaccinated and non-vaccinated coronavirus disease 2019 (COVID-19) patients are not well characterized. We sought to compare the clinical characteristics and host antiviral gene expression patterns of vaccinated and non-vaccinated cohorts at the Second People's Hospital of Fuyang City. METHODS: In this case-control study, we retrospectively analyzed 113 vaccinated patients with a COVID-19 Omicron variant infection, 46 non-vaccinated COVID-19 patients, and 24 healthy subjects (no history of COVID-19) recruited from the Second People's Hospital of Fuyang City. Blood samples were collected from each study participant for RNA extraction and PCR. We compared host antiviral gene expression profiles between healthy controls and COVID-19 patients who were either vaccinated or non-vaccinated at the time of infection. RESULTS: In the vaccinated group, most patients were asymptomatic, with only 42.9 % of patients developing fever. Notably, no patients had extrapulmonary organ damage. In contrast, 21.4 % of patients in the non-vaccinated group developed severe/critical (SC) disease and 78.6 % had mild/moderate (MM) disease, with fever occurring in 74.2 % patients. We found that Omicron infection in COVID-19 vaccinated patients was associated with significantly increased expression of several important host antiviral genes including IL12B, IL13, CXCL11, CXCL9, IFNA2, IFNA1, IFNγ, and TNFα. CONCLUSION: Vaccinated patients infected with the Omicron variant were mostly asymptomatic. In contrast, non-vaccinated patients frequently developed SC or MM disease. Older patients with SC COVID-19 also had a higher occurrence of mild liver dysfunction. Omicron infection in COVID-19 vaccinated patients was associated with the activation of key host antiviral genes and thus may play a role in reducing disease severity.
Subject(s)
Antiviral Agents , COVID-19 , Humans , Case-Control Studies , Retrospective Studies , COVID-19/epidemiology , SARS-CoV-2 , China/epidemiology , Vaccination , Disease Outbreaks , Fever , Gene ExpressionABSTRACT
Leucine-rich repeat kinase 2 (LRRK2) has been demonstrated to be closely involved in the pathogenesis of Parkinson's disease (PD), and pharmacological blockade of LRRK2 represents a new opportunity for therapeutical treatment of PD and other related neurodegenerative conditions. The development of an LRRK2-specific positron emission tomography (PET) ligand would enable a target occupancy study in vivo and greatly facilitate LRRK2 drug discovery and clinical translation as well as provide a molecular imaging tool for studying physiopathological changes in neurodegenerative diseases. In this work, we present the design and development of compound 8 (PF-06455943) as a promising PET radioligand through a PET-specific structure-activity relationship optimization, followed by comprehensive pharmacology and ADME/neuroPK characterization. Following an efficient 18F-labeling method, we have confirmed high brain penetration of [18F]8 in nonhuman primates (NHPs) and validated its specific binding in vitro by autoradiography in postmortem NHP brain tissues and in vivo by PET imaging studies.
Subject(s)
Parkinson Disease , Positron-Emission Tomography , Animals , Brain/diagnostic imaging , Brain/metabolism , Leucine/metabolism , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Ligands , Parkinson Disease/metabolism , Positron-Emission Tomography/methodsABSTRACT
Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18F-CHL-2205 (18F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.
Subject(s)
Alzheimer Disease , Brain , Alzheimer Disease/metabolism , Animals , Brain/metabolism , Cholesterol/metabolism , Cholesterol 24-Hydroxylase/metabolism , Female , Homeostasis , Humans , Male , Mammals/metabolism , MiceABSTRACT
The transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8) constitutes an auxiliary subunit of AMPA receptors, which mediates various brain functions including learning and memory. TARP γ-8 has emerged as a promising therapeutic target for central nervous system disorders. Despite considerable efforts, previously reported TARP γ-8 PET radioligands, such as [11C]TARP-1903 and [11C]TARP-1811 series, were plagued by limited brain uptake and/or high nonspecific binding in vivo. Herein, we developed two novel 11C-labeled probes, [11C]8 and [11C]15 (also named as [11C]TARP-2105), of which the latter exhibited a reasonable brain uptake as well as specific binding toward TARP γ-8 both in vitro and in vivo, as confirmed by blocking experiments with the commercially available TARP γ-8 inhibitor, JNJ-55511118 in the TARP γ-8-rich hippocampus. Overall, [11C]15 exhibited promising tracer characteristics and proved to be a lead positron-emission tomography ligand for the non-invasive quantification of TARP γ-8 in the mammalian brain.
Subject(s)
Calcium Channels , Receptors, AMPA , Animals , Brain/diagnostic imaging , Brain/metabolism , Calcium Channels/metabolism , Hippocampus/metabolism , Mammals/metabolism , Positron-Emission Tomography , Receptors, AMPA/metabolismABSTRACT
Monoacylglycerol lipase (MAGL) constitutes a serine hydrolase that orchestrates endocannabinoid homeostasis and exerts its function by catalyzing the degradation of 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA). As such, selective inhibition of MAGL represents a potential therapeutic and diagnostic approach to various pathologies including neurodegenerative disorders, metabolic diseases and cancers. Based on a unique 4-piperidinyl azetidine diamide scaffold, we developed a reversible and peripheral-specific radiofluorinated MAGL PET ligand [18F]FEPAD. Pharmacokinetics and binding studies on [18F]FEPAD revealed its outstanding specificity and selectivity towards MAGL in brown adipose tissue (BAT) - a tissue that is known to be metabolically active. We employed [18F]FEPAD in PET studies to assess the abundancy of MAGL in BAT deposits of mice and found a remarkable degree of specific tracer binding in the BAT, which was confirmed by post-mortem tissue analysis. Given the negative regulation of endocannabinoids on the metabolic BAT activity, our study supports the concept that dysregulation of MAGL is likely linked to metabolic disorders. Further, we now provide a suitable imaging tool that allows non-invasive assessment of MAGL in BAT deposits, thereby paving the way for detailed mechanistic studies on the role of BAT in endocannabinoid system (ECS)-related pathologies.
Subject(s)
Endocannabinoids , Monoacylglycerol Lipases , Endocannabinoids/metabolism , Adipose Tissue, Brown/diagnostic imaging , Adipose Tissue, Brown/metabolism , Positron-Emission Tomography/methods , Ligands , Enzyme Inhibitors/pharmacologyABSTRACT
Monoacylglycerol lipase (MAGL) is a pivotal enzyme in the endocannabinoid system, which metabolizes 2-arachidonoylglycerol (2-AG) into the proinflammatory eicosanoid precursor arachidonic acid (AA). MAGL and other endogenous cannabinoid (EC) degrading enzymes are involved in the fibrogenic signaling pathways that induce hepatic stellate cell (HSC) activation and ECM accumulation during chronic liver disease. Our group recently developed an 18F-labeled MAGL inhibitor ([18F]MAGL-4-11) for PET imaging and demonstrated highly specific binding in vitro and in vivo. In this study, we determined [18F]MAGL-4-11 PET enabled imaging MAGL levels in the bile duct ligation (BDL) and carbon tetrachloride (CCl4) models of liver cirrhosis; we also assessed the hepatic gene expression of the enzymes involved with EC system including MAGL, NAPE-PLD, FAAH and DAGL that as a function of disease severity in these models; [18F]MAGL-4-11 autoradiography was performed to assess tracer binding in frozen liver sections both in animal and human. [18F]MAGL-4-11 demonstrated reduced PET signals in early stages of fibrosis and further significantly decreased with disease progression compared with control mice. We confirmed MAGL and FAAH expression decreases with fibrosis severity, while its levels in normal liver tissue are high; in contrast, the EC synthetic enzymes NAPE-PLD and DAGL are enhanced in these different fibrosis models. In vitro autoradiography further supported that [18F]MAGL-4-11 bound specifically to MAGL in both animal and human fibrotic liver tissues. Our PET ligand [18F]MAGL-4-11 shows excellent sensitivity and specificity for MAGL visualization in vivo and accurately reflects the histological stages of liver fibrosis in preclinical models and human liver tissues.
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Hepatocellular carcinoma (HCC) is a prevalent liver malignancy, which ranks third in the cancer-related cause of deaths in worldwide and ninth in the United States. Currently, HCC is typically diagnosed by ultrasound, computed tomography (CT) and magnetic resonance imaging (MRI) scan at its late stage and the survival of HCC patients after diagnosis is usually very poor. Therefore, the development of novel and effective tool for early diagnosis, characterization and staging of HCC patients is of critical importance. Recent studies have demonstrated correlation of HCC with MAGL. In HCC cells, upregulation of MAGL activity enhanced cell invasiveness ability, while pharmacological blockade of MAGL led to significant inhibition of this trend. In this study, we aim to visualize the expression and activity of hepatic MAGL in different HCC cells and HCC patients' samples by taking advantage of positron emission tomography (PET) imaging with our previously developed MAGL radioligand [11C]MAGL-0519. As a result, [11C]MAGL-0519 exhibited higher radioactivity accumulation in HepaG2 and Hepa 1-6 cell lines compared with that of normal liver cells (AML-12 and LX-2), indicating higher MAGL expression levels in these HCC cells. This rationale was then validated by Western blot and immunofluorescent staining analysis. Furthermore, HCC patients' liver sections exhibited significantly increased uptake of [11C]MAGL-0519, which was consistent with the results in cell uptake assays. Taking together, these results provided a biological rationale and built a foundation to use [11C]MAGL-0519 as a potential and effective PET ligand for the diagnosis of HCC.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Carcinoma, Hepatocellular/diagnostic imaging , Carcinoma, Hepatocellular/pathology , Humans , Ligands , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/pathology , Positron-Emission TomographyABSTRACT
Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.
Subject(s)
Hepatitis B/pathology , Hepatitis C/pathology , Liver Cirrhosis/pathology , Nanog Homeobox Protein/metabolism , Octamer Transcription Factor-3/metabolism , Transforming Growth Factor beta1/metabolism , Actins/biosynthesis , Adult , CRISPR-Cas Systems/genetics , Cell Line, Tumor , Cell Movement/physiology , Coinfection/pathology , Collagen Type I, alpha 1 Chain/biosynthesis , Female , Gene Knockout Techniques , Hepacivirus/metabolism , Hepatic Stellate Cells/pathology , Hepatic Stellate Cells/virology , Hepatitis B virus/metabolism , Hepatocytes/pathology , Hepatocytes/virology , Humans , Liver/pathology , Liver Cirrhosis/virology , Male , Nanog Homeobox Protein/genetics , Octamer Transcription Factor-3/genetics , Organic Anion Transporters, Sodium-Dependent/metabolism , Symporters/metabolism , Tissue Inhibitor of Metalloproteinase-1/biosynthesisABSTRACT
Background: Esophageal variceal (EV) hemorrhage is a life-threatening consequence of portal hypertension in cirrhotic patients. Screening upper endoscopy and endoscopic variceal ligation to identify and treat EVs have contraindications, complications, and high costs. We sought to identify non-invasive tests (NITs) as alternatives to endoscopic EV screening. Methods: In this case-control study, we retrospectively analyzed 286 cirrhotic patients treated for EVs at the Second People's Hospital of Fuyang City, China from January to December 2019. We applied ROC curve analysis to assess the accuracy of various NITs in predicting EV hemorrhage. Results: There were significant differences between the hemorrhage and non-hemorrhage groups in median serum albumin (ALB) (p < 0.001), median bilirubin (TBIL) (p < 0.046), prothrombin (PT) time (p < 0.001), Golgi protein 73 (GP73; p = 0.012) and Child-Pugh (C-P) scores (p < 0.001). For ALB (cutoff <33.2g/L), PT time (cutoff > 14.2 seconds), GP73 (cutoff > 126.4 ng/ml), and C-P scores, the areas under the ROC curves (AUCs) were 73.4% (95% CI: 67.5-79.2), 68.6% (95% CI: 62.4-74.8), 62.2% (95% CI: 52.8-71.5) and 69.8% (95%CI: 63.8-75.8), respectively, with corresponding sensitives of 71.5, 59.8, 69.8, and 92.2% and specificities of 65.6%, 70.1%, 56.5%, and 38.6%. When ALB was combined with GP73, the AUC was 74.3% (95% CI: 66.1-82.5) with a sensitivity of 65.1% and specificity of 76.5%. When ALB, PT, and C-P scores were combined, the AUC was 76.5% (95% CI: 70.9-82.1) with a sensitivity of 79.5% and specificity of 64.3%. When ALB, PT, GP73, and C-P scores were combined, the AUC was 75.2% (95% CI: 67.3-83.1) with a sensitivity of 54.0% and specificity of 86.9%. Conclusion: ALB, TBIL, GP73, and C-P scores, may be used to predict EV hemorrhage in cirrhotic patients. The combination of multiple NITs is better than a single index and can increase diagnostic performance.
ABSTRACT
Autotaxin (ATX) is a secreted phosphodiesterase that has been implicated in a remarkably wide array of pathologies, especially in fibrosis and cancer. While ATX inhibitors have entered the clinical arena, a validated probe for positron emission tomography (PET) is currently lacking. With the aim to develop a suitable ATX-targeted PET radioligand, we have synthesized a focused library of fluorinated imidazo[1,2-a]pyridine derivatives, determined their inhibition constants, and confirmed their binding mode by crystallographic analysis. Based on their promising in vitro properties, compounds 9c, 9f, 9h, and 9j were radiofluorinated. Also, a deuterated analog of [18F]9j, designated as [18F]ATX-1905 ([18F]20), was designed and proved to be highly stable against in vivo radiodefluorination compared with [18F]9c, [18F]9f, [18F]9h, and [18F]9j. These results along with in vitro and in vivo studies toward ATX in a mouse model of LPS-induced liver injury suggest that [18F]ATX-1905 is a suitable PET probe for the non-invasive quantification of ATX.
Subject(s)
Enzyme Inhibitors/pharmacology , Phosphoric Diester Hydrolases/analysis , Positron-Emission Tomography , Radiopharmaceuticals/pharmacology , Animals , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemistry , Fluorine Radioisotopes , Ligands , Mice , Molecular Structure , Phosphoric Diester Hydrolases/metabolism , Radiopharmaceuticals/chemistry , Structure-Activity RelationshipABSTRACT
OBJECTIVES: To enable non-invasive real-time quantification of vasopressin 1A (V1A) receptors in peripheral organs, we sought to develop a suitable PET probe that would allow specific and selective V1A receptor imaging in vitro and in vivo. METHODS: We synthesized a high-affinity and -selectivity ligand, designated compound 17. The target structure was labeled with carbon-11 and tested for its utility as a V1A-targeted PET tracer by cell uptake studies, autoradiography, in vivo PET imaging and ex vivo biodistribution experiments. RESULTS: Compound 17 (PF-184563) and the respective precursor for radiolabeling were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. An inhibitory constant of 0.9 nM towards the V1A receptors was measured, while excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM) were obtained. Cell uptake studies revealed considerable V1A binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. In vitro autoradiography and PET imaging studies in rodents indicated specific tracer binding mainly in the liver. Further, the pancreas, spleen and the heart exhibited specific binding of [11C]17 ([11C]PF-184563) by ex vivo biodistribution experiments. CONCLUSION: We have developed the first V1A-targeted PET ligand that is suitable for subtype-selective receptor imaging in peripheral organs including the liver, heart, pancreas and spleen. Our findings suggest that [11C]PF-184563 can be a valuable tool to study the role of V1A receptors in liver diseases, as well as in cardiovascular pathologies.