Publications by authors named "Jifeng Zhang"

215 Publications

Untargeted metabolomics identifies succinate as a biomarker and therapeutic target in aortic aneurysm and dissection.

Eur Heart J 2021 Sep 17. Epub 2021 Sep 17.

The Institute of Cardiovascular Sciences and Institute of Systems Biomedicine, School of Basic Medical Sciences, Key Laboratory of Molecular Cardiovascular Sciences of Ministry of Education, Health Sciences Center, Peking University, Xueyuan Road NO.38, Haidian District, Beijing 100871, China.

Aims: Aortic aneurysm and dissection (AAD) are high-risk cardiovascular diseases with no effective cure. Macrophages play an important role in the development of AAD. As succinate triggers inflammatory changes in macrophages, we investigated the significance of succinate in the pathogenesis of AAD and its clinical relevance.

Methods And Results: We used untargeted metabolomics and mass spectrometry to determine plasma succinate concentrations in 40 and 1665 individuals of the discovery and validation cohorts, respectively. Three different murine AAD models were used to determine the role of succinate in AAD development. We further examined the role of oxoglutarate dehydrogenase (OGDH) and its transcription factor cyclic adenosine monophosphate-responsive element-binding protein 1 (CREB) in the context of macrophage-mediated inflammation and established p38αMKOApoe-/- mice. Succinate was the most upregulated metabolite in the discovery cohort; this was confirmed in the validation cohort. Plasma succinate concentrations were higher in patients with AAD compared with those in healthy controls, patients with acute myocardial infarction (AMI), and patients with pulmonary embolism (PE). Moreover, succinate administration aggravated angiotensin II-induced AAD and vascular inflammation in mice. In contrast, knockdown of OGDH reduced the expression of inflammatory factors in macrophages. The conditional deletion of p38α decreased CREB phosphorylation, OGDH expression, and succinate concentrations. Conditional deletion of p38α in macrophages reduced angiotensin II-induced AAD.

Conclusion: Plasma succinate concentrations allow to distinguish patients with AAD from both healthy controls and patients with AMI or PE. Succinate concentrations are regulated by the p38α-CREB-OGDH axis in macrophages.
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http://dx.doi.org/10.1093/eurheartj/ehab605DOI Listing
September 2021

Inhibition of a Novel CLK1-THRAP3-PPARγ Axis Improves Insulin Sensitivity.

Front Physiol 2021 30;12:699578. Epub 2021 Aug 30.

Department of Internal Medicine, Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, MI, United States.

Increasing energy expenditure by promoting "browning" in adipose tissues is a promising strategy to prevent obesity and associated diabetes. To uncover potential targets of cold exposure, which induces energy expenditure, we performed phosphoproteomics profiling in brown adipose tissue of mice housed in mild cold environment at 16°C. We identified CDC2-like kinase 1 (CLK1) as one of the kinases that were significantly downregulated by mild cold exposure. In addition, genetic knockout of CLK1 or chemical inhibition in mice ameliorated diet-induced obesity and insulin resistance at 22°C. Through proteomics, we uncovered thyroid hormone receptor-associated protein 3 (THRAP3) as an interacting partner of CLK1, further confirmed by co-immunoprecipitation assays. We further demonstrated that CLK1 phosphorylates THRAP3 at Ser243, which is required for its regulatory interaction with phosphorylated peroxisome proliferator-activated receptor gamma (PPARγ), resulting in impaired adipose tissue browning and insulin sensitivity. These data suggest that CLK1 plays a critical role in controlling energy expenditure through the CLK1-THRAP3-PPARγ axis.
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http://dx.doi.org/10.3389/fphys.2021.699578DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8435799PMC
August 2021

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression.

Thromb Haemost 2021 Aug 24. Epub 2021 Aug 24.

Department of Internal Medicine, Frankel Cardiovascular Center, University of Michigan Medical Center, Ann Arbor, Michigan, United States.

Krüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor () gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to gene promoter in ECs. In addition, we demonstrated that conventional knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating thrombosis.
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http://dx.doi.org/10.1055/s-0041-1735191DOI Listing
August 2021

hiPSC Modeling of Lineage-Specific Smooth Muscle Cell Defects Caused by Variant, and its Therapeutic Implications for Loeys-Dietz Syndrome.

Circulation 2021 Aug 4. Epub 2021 Aug 4.

Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI.

Loeys-Dietz Syndrome (LDS) is an inherited disorder predisposing individuals to thoracic aortic aneurysm and dissection (TAAD). Currently, there are no medical treatments except surgical resection. Although the genetic basis of LDS is well-understood, molecular mechanisms underlying the disease remain elusive impeding the development of a therapeutic strategy. In addition, aortic smooth muscle cells (SMC) have heterogenous embryonic origins depending on their spatial location, and lineage-specific effects of pathogenic variants on SMC function, likely causing regionally constrained LDS manifestations, have been unexplored. We identified an LDS family with a dominant pathogenic variant in gene () causing aortic root aneurysm and dissection. To accurately model the molecular defects caused by this mutation, we used human-induced pluripotent stem cells (hiPSC) from subject with normal aorta to generate hiPSC carrying , and corrected the mutation in patient-derived hiPSC using CRISPR-Cas9 gene editing. Following their lineage-specific SMC differentiation through cardiovascular progenitor cell (CPC) and neural crest stem cell (NCSC) lineages, we employed conventional molecular techniques and single-cell RNA-sequencing (scRNA-seq) to characterize the molecular defects. The resulting data led to subsequent molecular and functional rescue experiments employing Activin A and rapamycin. Our results indicate the mutation impairs contractile transcript and protein levels, and function in CPC-SMC, but not in NCSC-SMC. ScRNA-seq results implicate defective differentiation even in CPC-SMC including disruption of SMC contraction, and extracellular matrix formation. Comparison of patient-derived and mutation-corrected cells supported the contractile phenotype observed in the mutant CPC-SMC. selectively disrupted SMAD3 and AKT activation in CPC-SMC, and led to increased cell proliferation. Consistently, scRNA-seq revealed molecular similarities between a loss-of-function SMAD3 mutation () and . Lastly, combination treatment with Activin A and rapamycin during or after SMC differentiation significantly improved the mutant CPC-SMC contractile gene expression, and function; and rescued the mechanical properties of mutant CPC-SMC tissue constructs. This study reveals that a pathogenic variant causes lineage-specific SMC defects informing the etiology of LDS-associated aortic root aneurysm. As a potential pharmacological strategy, our results highlight a combination treatment with Activin A and rapamycin that can rescue the SMC defects caused by the variant.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.121.054744DOI Listing
August 2021

Dysregulated oxalate metabolism is a driver and therapeutic target in atherosclerosis.

Cell Rep 2021 Jul;36(4):109420

Department of Internal Medicine, Frankel Cardiovascular Center, University of Michigan, Ann Arbor, MI 48109, USA; Department of Pathology and Translational Pathobiology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA 71103, USA. Electronic address:

Dysregulated glycine metabolism is emerging as a common denominator in cardiometabolic diseases, but its contribution to atherosclerosis remains unclear. In this study, we demonstrate impaired glycine-oxalate metabolism through alanine-glyoxylate aminotransferase (AGXT) in atherosclerosis. As found in patients with atherosclerosis, the glycine/oxalate ratio is decreased in atherosclerotic mice concomitant with suppression of AGXT. Agxt deletion in apolipoprotein E-deficient (Apoe) mice decreases the glycine/oxalate ratio and increases atherosclerosis with induction of hepatic pro-atherogenic pathways, predominantly cytokine/chemokine signaling and dysregulated redox homeostasis. Consistently, circulating and aortic C-C motif chemokine ligand 5 (CCL5) and superoxide in lesional macrophages are increased. Similar findings are observed following dietary oxalate overload in Apoe mice. In macrophages, oxalate induces mitochondrial dysfunction and superoxide accumulation, leading to increased CCL5. Conversely, AGXT overexpression in Apoe mice increases the glycine/oxalate ratio and decreases aortic superoxide, CCL5, and atherosclerosis. Our findings uncover dysregulated oxalate metabolism via suppressed AGXT as a driver and therapeutic target in atherosclerosis.
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http://dx.doi.org/10.1016/j.celrep.2021.109420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8363062PMC
July 2021

Regulatory variants in TCF7L2 are associated with thoracic aortic aneurysm.

Am J Hum Genet 2021 09 14;108(9):1578-1589. Epub 2021 Jul 14.

K.G. Jebsen Center for Genetic Epidemiology, Department of Public Health and Nursing, NTNU, Norwegian University of Science and Technology, Trondheim 7030, Norway.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.016DOI Listing
September 2021

Neoadjuvant chemoradiotherapy plus postoperative adjuvant XELOX chemotherapy versus postoperative adjuvant chemotherapy with XELOX regimen for local advanced gastric cancer-A randomized, controlled study.

Br J Radiol 2021 Aug 14;94(1124):20201088. Epub 2021 Jul 14.

Department of Oncology, Shandong Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jinan, China.

Objective: The aim of this study was to compare the clinical efficacy of neoadjuvant chemoradiotherapy (NACRT) combined with postoperative adjuvant XELOX (Oxaliplatin +Capecitabine) chemotherapy and postoperative adjuvant chemotherapy (ACT) with XELOX for local advanced gastric cancer (LAGC).

Methods: In this prospectively randomized trial, we investigated the effect of NACRT combined with postoperative ACT for LAGC. 60 patients were randomly divided into NACRT group and ACT group, with 30 patients in each group. Patients in NACRT group were given three-dimensional conformal radiotherapy (45 Gy/1.8 Gy/f) accompanied by synchronous XELOX of two cycles, followed by surgery, and then postoperative adjuvant XELOX chemotherapy of four cycles was performed. Patients in ACT group received surgery in advance, and then XELOX chemotherapy of six cycles was given.

Results: The objective response rate of NACRT was 76.7%. The overall incidence of postoperative complications in NACRT group was not significantly different from that in ACT group (23.1% 30.0%, = 0.560). The 1 year, 2 years, and 3 years progression-free survival (PFS)and overall survival (OS) in NACRT and ACT groups were 80.0% 56.7%, 73.3% 46.7%, 60.0% 33.3%, and 86.7% 80.0%, 76.7% 66.7%, 63.3% 50.0%, respectively. Patients in NACRT group showed a significantly higher R0 resection rate (84.6% 56.7%, = 0.029),lower loco-regional recurrence rate (36.7% 11.5%, = 0.039), longer PFS ( = 0.019) and freedom from locoregional progression(FFLP) ( = 0.004) than patients in ACT group, while there was no difference in OS ( = 0.215) and in toxicity incidence ( > 0.05).

Conclusions: NACRT combined with postoperative adjuvant XELOX chemotherapy can improve R0 resection rate, reduce loco-regional recurrence, prolong PFS and FFLP without increasing the incidence of postoperative complications in patients with LAGC.

Advances In Knowledge: Compared with postoperative adjuvant chemotherapy, locally advanced gastric cancer patients may benefit from neoadjuvant chemoradiotherapy, and toxicity associated with chemoradiotherapy was tolerant and manageable.
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http://dx.doi.org/10.1259/bjr.20201088DOI Listing
August 2021

Cancer spheroids derived exosomes reveal more molecular features relevant to progressed cancer.

Biochem Biophys Rep 2021 Jul 25;26:101026. Epub 2021 May 25.

School of Biological Engineering, Huainan Normal University, Huainan, Anhui, 232038, China.

Cancer cell spheroids have been shown to be more physiologically relevant to native tumor tissue than monolayer 2D culture cells. Due to enhanced intercellular communications among cells in spheroids, spheroid secreted exosomes which account for transcellular transportation should exceed those from 2D cell culture and may display a different expression pattern of miRNA or protein. To test this, we employed a widely used pancreatic cancer cell line, PANC-1, to create 3D spheroids and compared exosomes generated by both 2D cell culture and 3D PANC-1 spheroids. We further measured and compared exosomal miRNA and GPC-1 protein expression with qRT-PCR and enzyme-linked immunosorbent assay, respectively. It showed that PANC-1 cells cultured in 3D spheroids can produce significantly more exosomes than PANC-1 2D cells and exosomal miRNA and GPC-1 expression derived from spheroids show more features relevant to the progression of pancreatic cancer. These findings point to the potential importance of using spheroids as model to study cancer development and progression.
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http://dx.doi.org/10.1016/j.bbrep.2021.101026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8167213PMC
July 2021

Single-Cell Transcriptomics Reveals Endothelial Plasticity During Diabetic Atherogenesis.

Front Cell Dev Biol 2021 19;9:689469. Epub 2021 May 19.

Frankel Cardiovascular Center, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, United States.

Atherosclerosis is the leading cause of cardiovascular diseases, which is also the primary cause of mortality among diabetic patients. Endothelial cell (EC) dysfunction is a critical early step in the development of atherosclerosis and aggravated in the presence of concurrent diabetes. Although the heterogeneity of the organ-specific ECs has been systematically analyzed at the single-cell level in healthy conditions, their transcriptomic changes in diabetic atherosclerosis remain largely unexplored. Here, we carried out a single-cell RNA sequencing (scRNA-seq) study using EC-enriched single cells from mouse heart and aorta after 12 weeks feeding of a standard chow or a diabetogenic high-fat diet with cholesterol. We identified eight EC clusters, three of which expressed mesenchymal markers, indicative of an endothelial-to-mesenchymal transition (EndMT). Analyses of the marker genes, pathways, and biological functions revealed that ECs are highly heterogeneous and plastic both in normal and atherosclerotic conditions. The metabolic transcriptomic analysis further confirmed that EndMT-derived fibroblast-like cells are prominent in atherosclerosis, with diminished fatty acid oxidation and enhanced biological functions, including regulation of extracellular-matrix organization and apoptosis. In summary, our data characterized the phenotypic and metabolic heterogeneity of ECs in diabetes-associated atherogenesis at the single-cell level and paves the way for a deeper understanding of endothelial cell biology and EC-related cardiovascular diseases.
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http://dx.doi.org/10.3389/fcell.2021.689469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8170046PMC
May 2021

Improving the genome assembly of rabbits with long-read sequencing.

Genomics 2021 Sep 27;113(5):3216-3223. Epub 2021 May 27.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, Ann Arbor, MI, USA. Electronic address:

The European rabbit (Oryctolagus cuniculus) is important as a biomedical model given its unique features in immunity and metabolism. The current reference genome OryCun2.0 established with whole-genome shotgun sequencing was quite fragmented and had not been updated for ten years. In this work, we provided a new rabbit genome assembly UM_NZW_1.0 to improve OryCun2.0 by leveraging the contig lengths based on long-read sequencing and a wealth of available Illumina paired-end sequence data. UM_NZW_1.0 showed a remarkable increase of continuity compared with OryCun2.0, with 5 times longer contig N50 and approximately 75% gaps closed. Many of the closed gaps were overlapped with protein-coding genes or transcriptional features, resulting in an enhancement of gene annotations. In particular, UM_NZW_1.0 presented a more complete landscape of the MHC region and the IGH locus, therefore provided a valuable resource for future researches on rabbits.
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http://dx.doi.org/10.1016/j.ygeno.2021.05.031DOI Listing
September 2021

Integration of Transformative Platforms for the Discovery of Causative Genes in Cardiovascular Diseases.

Cardiovasc Drugs Ther 2021 06 15;35(3):637-654. Epub 2021 Apr 15.

Department of Internal Medicine, University of Michigan Medical Center, 2800 Plymouth Rd, Ann Arbor, MI, 48109-2800, USA.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Genome-wide association studies (GWAS) are powerful epidemiological tools to find genes and variants associated with cardiovascular diseases while follow-up biological studies allow to better understand the etiology and mechanisms of disease and assign causality. Improved methodologies and reduced costs have allowed wider use of bulk and single-cell RNA sequencing, human-induced pluripotent stem cells, organoids, metabolomics, epigenomics, and novel animal models in conjunction with GWAS. In this review, we feature recent advancements relevant to cardiovascular diseases arising from the integration of genetic findings with multiple enabling technologies within multidisciplinary teams to highlight the solidifying transformative potential of this approach. Well-designed workflows integrating different platforms are greatly improving and accelerating the unraveling and understanding of complex disease processes while promoting an effective way to find better drug targets, improve drug design and repurposing, and provide insight towards a more personalized clinical practice.
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http://dx.doi.org/10.1007/s10557-021-07175-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216854PMC
June 2021

A Novel Dual Drug Approach That Combines Ivermectin and Dihydromyricetin (DHM) to Reduce Alcohol Drinking and Preference in Mice.

Molecules 2021 Mar 22;26(6). Epub 2021 Mar 22.

Titus Family Department of Clinical Pharmacy, University of Southern California School of Pharmacy, Los Angeles, CA 90089, USA.

Alcohol use disorder (AUD) affects over 18 million people in the US. Unfortunately, pharmacotherapies available for AUD have limited clinical success and are under prescribed. Previously, we established that avermectin compounds (ivermectin [IVM] and moxidectin) reduce alcohol (ethanol/EtOH) consumption in mice, but these effects are limited by P-glycoprotein (Pgp/ABCB1) efflux. The current study tested the hypothesis that dihydromyricetin (DHM), a natural product suggested to inhibit Pgp, will enhance IVM potency as measured by changes in EtOH consumption. Using a within-subjects study design and two-bottle choice study, we tested the combination of DHM (10 mg/kg; i.p.) and IVM (0.5-2.5 mg/kg; i.p.) on EtOH intake and preference in male and female C57BL/6J mice. We also conducted molecular modeling studies of DHM with the nucleotide-binding domain of human Pgp that identified key binding residues associated with Pgp inhibition. We found that DHM increased the potency of IVM in reducing EtOH consumption, resulting in significant effects at the 1.0 mg/kg dose. This combination supports our hypothesis that inhibiting Pgp improves the potency of IVM in reducing EtOH consumption. Collectively, we demonstrate the feasibility of this novel combinatorial approach in reducing EtOH consumption and illustrate the utility of DHM in a novel combinatorial approach.
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http://dx.doi.org/10.3390/molecules26061791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8004700PMC
March 2021

Nano-selenium controlled cadmium accumulation and improved photosynthesis in indica rice cultivated in lead and cadmium combined paddy soils.

J Environ Sci (China) 2021 May 8;103:336-346. Epub 2020 Dec 8.

School of Biological Engineering, Huainan Normal University, Huainan 232038, China; Key Laboratory of Bioresource and Environmental Biotechnology of Anhui Higher Education Institutes, Huainan Normal University, Huainan 232038, China.

Selenium nanoparticles (Se NPs) are less toxic and more biocompatible than selenite or selenate. However, studies involving spraying with Se NPs for reducing accumulation of cadmium (Cd) and lead (Pb) in rice grains have been rarely reported as yet. Herein, indica rice seedlings cultivated in Cd+Pb-spiked paddy soils (denoted as positive control) were sprayed with Se NPs sols for four times from tillering to booting stage. Compared to positive control, 50-100 μmol/L Se NPs downregulated Cd transporters-related genes such as OsLCT1, OsHMA2 and OsCCX2 in leaves and OsLCT1, OsPCR1 and OsCCX2 genes in node I at filling stage. Meanwhile, Se-binding protein 1 was distinctly elevated, involving the repression of Cd and Pb transportation to rice grains. Se NPs also differentially improved RuBP carboxylase and chlorophylls especially some key genes and proteins involving photosynthetic system. Besides, 25-50 μmol/L Se NPs diminished reactive oxygen species overproduction from NADPH oxidases whereas boosted glutathione peroxidase, reducing protein carbonylation in rice seedlings. However, the antioxidant isozymes and oxidatively modified proteins were slightly rebounded at 100 μmol/L. Se contents were noticeably elevated and confirmed to exist as selenomethionine in the rice grains following all the treatments by Se NPs. Thus, the optimal dosage of Se NPs for foliar application is 50 μmol/L, which significantly decreased Cd accumulation, improved photosynthesis and Se enrichment whereas caused no distinct reduction of Pb in the grains. Thus, an appropriate dosage of Se NPs can be conducted to decrease Cd accumulation, improve photosynthesis, and organic Se contents in rice grains.
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http://dx.doi.org/10.1016/j.jes.2020.11.005DOI Listing
May 2021

Development of the Nude Rabbit Model.

Stem Cell Reports 2021 03 18;16(3):656-665. Epub 2021 Feb 18.

Center for Advanced Models and Translational Sciences and Therapeutics, University of Michigan, Ann Arbor, MI 48109, USA. Electronic address:

Loss-of-function mutations in the forkhead box N1 (FOXN1) gene lead to nude severe combined immunodeficiency, a rare inherited syndrome characterized by athymia, severe T cell immunodeficiency, congenital alopecia, and nail dystrophy. We recently produced FOXN1 mutant nude rabbits (NuRabbits) by using CRISPR-Cas9. Here we report the establishment and maintenance of the NuRabbit colony. NuRabbits, like nude mice, are hairless, lack thymic development, and are immunodeficient. To demonstrate the functional applications of NuRabbits in biomedical research, we show that they can successfully serve as the recipient animals in xenotransplantation experiments using human induced pluripotent stem cells or tissue-engineered blood vessels. Our work presents the NuRabbit as a new member of the immunodeficient animal model family. The relatively large size and long lifespan of NuRabbits offer unique applications in regenerative medicine, cancer research, and the study of a variety of other human conditions, including immunodeficiency.
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http://dx.doi.org/10.1016/j.stemcr.2021.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940256PMC
March 2021

Gene Editing in Rabbits: Unique Opportunities for Translational Biomedical Research.

Front Genet 2021 28;12:642444. Epub 2021 Jan 28.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, Ann Arbor, MI, United States.

The rabbit is a classic animal model for biomedical research, but the production of gene targeted transgenic rabbits had been extremely challenging until the recent advent of gene editing tools. More than fifty gene knockout or knock-in rabbit models have been reported in the past decade. Gene edited (GE) rabbit models, compared to their counterpart mouse models, may offer unique opportunities in translational biomedical research attributed primarily to their relatively large size and long lifespan. More importantly, GE rabbit models have been found to mimic several disease pathologies better than their mouse counterparts particularly in fields focused on genetically inherited diseases, cardiovascular diseases, ocular diseases, and others. In this review we present selected examples of research areas where GE rabbit models are expected to make immediate contributions to the understanding of the pathophysiology of human disease, and support the development of novel therapeutics.
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http://dx.doi.org/10.3389/fgene.2021.642444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876448PMC
January 2021

Quantification and evolution of mitochondrial genome rearrangement in Amphibians.

BMC Ecol Evol 2021 02 9;21(1):19. Epub 2021 Feb 9.

College of Life Science, Anhui Normal University, Wuhu, Anhui, 241000, People's Republic of China.

Background: Rearrangement is an important topic in the research of amphibian mitochondrial genomes ("mitogenomes" hereafter), whose causes and mechanisms remain enigmatic. Globally examining mitogenome rearrangements and uncovering their characteristics can contribute to a better understanding of mitogenome evolution.

Results: Here we systematically investigated mitogenome arrangements of 232 amphibians including four newly sequenced Dicroglossidae mitogenomes. The results showed that our new sequenced mitogenomes all possessed a trnM tandem duplication, which was not exclusive to Dicroglossidae. By merging the same arrangements, the mitogenomes of ~ 80% species belonged to the four major patterns, the major two of which were typical vertebrate arrangement and typical neobatrachian arrangement. Using qMGR for calculating rearrangement frequency (RF) (%), we found that the control region (CR) (RF = 45.04) and trnL2 (RF = 38.79) were the two most frequently rearranged components. Forty-seven point eight percentage of amphibians possessed rearranged mitogenomes including all neobatrachians and their distribution was significantly clustered in the phylogenetic trees (p < 0.001). In addition, we argued that the typical neobatrachian arrangement may have appeared in the Late Jurassic according to possible occurrence time estimation.

Conclusion: It was the first global census of amphibian mitogenome arrangements from the perspective of quantity statistics, which helped us to systematically understand the type, distribution, frequency and phylogenetic characteristics of these rearrangements.
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http://dx.doi.org/10.1186/s12862-021-01755-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7871395PMC
February 2021

Production of CFTR-ΔF508 Rabbits.

Front Genet 2020 22;11:627666. Epub 2021 Jan 22.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical Center, University of Michigan Medical School, Ann Arbor, MI, United States.

Cystic Fibrosis (CF) is a lethal autosomal recessive disease caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation is the deletion of phenylalanine residue at position 508 (ΔF508). Here we report the production of CFTR-ΔF508 rabbits by CRISPR/Cas9-mediated gene editing. After microinjection and embryo transfer, 77 kits were born, of which five carried the ΔF508 mutation. To confirm the germline transmission, one male ΔF508 founder was bred with two wild-type females and produced 16 F1 generation kits, of which six are heterozygous ΔF508/WT animals. Our work adds CFTR-ΔF508 rabbits to the toolbox of CF animal models for biomedical research.
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http://dx.doi.org/10.3389/fgene.2020.627666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862758PMC
January 2021

Krüppel-Like Factor 14 Deletion In Myeloid Cells Accelerates Atherosclerotic Lesion Development.

Cardiovasc Res 2021 Feb 4. Epub 2021 Feb 4.

Molecular and Integrative Physiology, University of Michigan Medical Center, Ann Arbor, Michigan, USA.

Aims: Atherosclerosis is the dominant pathologic basis of many cardiovascular diseases. Large genome-wide association studies have identified that single nucleotide polymorphisms proximal to Krüppel-like factor 14 (KLF14), a member of the zinc finger family of transcription factors, are associated with higher cardiovascular risks. Macrophage dysfunction contributes to atherosclerosis development and has been recognized as a potential therapeutic target for treating many cardiovascular diseases. Herein, we address the biologic function of KLF14 in macrophages and its role during the development of atherosclerosis.

Methods And Results: KLF14 expression was markedly decreased in cholesterol-loaded foam cells, and overexpression of KLF14 significantly increased cholesterol efflux and inhibited the inflammatory response in macrophages. We generated myeloid cell-selective Klf14 knockout (Klf14LysM) mice in the ApoE-/- background for the atherosclerosis study. Klf14LysMApoE-/- and litter-mate control mice (Klf14fl/flApoE-/-) were placed on the Western Diet for 12 weeks to induce atherosclerosis. Macrophage Klf14 deficiency resulted in increased atherosclerosis development without affecting the plasma lipid profiles. Klf14-deficient peritoneal macrophages showed significantly reduced cholesterol efflux resulting in increased lipid accumulation and exacerbated inflammatory response. Mechanistically, KLF14 upregulates the expression of a key cholesterol efflux transporter, ABCA1 (ATP-binding cassette transporter A1), while suppresses the expression of several critical components of the inflammatory cascade. In macrophages, activation of KLF14 by its activator, perhexiline, a drug clinically used to treat angina, significantly inhibited the inflammatory response and increased cholesterol efflux in a KLF14- dependent manner in macrophages without triggering hepatic lipogenesis.

Conclusions: This study provides insights into the anti-atherosclerotic effects of macrophage KLF14 through promoting cholesterol efflux and suppressing the inflammatory response. Activation of KLF14 may represent a potential new therapeutic approach to prevent or treat atherosclerosis.

Translational Perspective: Here, using both gain- and loss-of-function strategies, we show that KLF14 regulates cholesterol efflux by regulating the expression of ABCA1 and inhibits inflammatory response in macrophages. These findings, along with our previous data, put activation of KLF14 forward as a prospective therapeutic target for atherosclerotic cardiovascular disease.
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http://dx.doi.org/10.1093/cvr/cvab027DOI Listing
February 2021

New Insight Into Metformin-Induced Cholesterol-Lowering Effect Crosstalk Between Glucose and Cholesterol Homeostasis via ChREBP (Carbohydrate-Responsive Element-Binding Protein)-Mediated PCSK9 (Proprotein Convertase Subtilisin/Kexin Type 9) Regulation.

Arterioscler Thromb Vasc Biol 2021 04 4;41(4):e208-e223. Epub 2021 Feb 4.

Department of Cardiovascular Medicine, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China (D.H., J.L., B.Y., Y.L., D.P.).

[Figure: see text].
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http://dx.doi.org/10.1161/ATVBAHA.120.315708DOI Listing
April 2021

CRMarker: A manually curated comprehensive resource of cancer RNA markers.

Int J Biol Macromol 2021 Mar 30;174:263-269. Epub 2021 Jan 30.

Department of Biostatistics and Computational Biology, School of Life Sciences, Fudan University, Shanghai 200436, PR China. Electronic address:

Biomolecular markers have extremely important value for cancer research and treatment. However, as far as we know, there are still no searchable and predictable resources focusing on multiple classes of RNA molecular markers in cancers. Herein, we developed CRMarker, a manually curated comprehensive repository of cancer RNA markers. In the current release, CRMarker v1.1 consists of 5489 "known" cancer RNA markers based on 8756 valid publications in PubMed, including 2878 mRNAs (genes), 1314 miRNAs, 1097 lncRNAs and 200 circRNAs, and involving two functional molecules (diagnosis and prognosis), 21 organisms and 154 cancers. The search results provided by the database are comprehensive, including 11 items such as RNA molecule expression and risk level, type of tissue or sample, cancer subtype, reference type, etc. Moreover, CRMarker also provides more than 18,000 potential cancer RNA markers, which are predicted based on "guilt-by-association" analysis of the above-mentioned "known" RNA markers and three molecular interaction networks, and survival analysis of 18 gene expression data sets with survival data. CRMarker v1.1 has a friendly interface and is freely available online at http://crmarker.hnnu.edu.cn/. We aim to build a comprehensive platform that is convenient for cancer researchers and clinicians to inquire and retrieve.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.01.186DOI Listing
March 2021

KLF11 protects against abdominal aortic aneurysm through inhibition of endothelial cell dysfunction.

JCI Insight 2021 03 8;6(5). Epub 2021 Mar 8.

Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, USA.

Abdominal aortic aneurysm (AAA) is a life-threatening degenerative vascular disease. Endothelial cell (EC) dysfunction is implicated in AAA. Our group recently demonstrated that Krüppel-like factor 11 (KLF11) plays an essential role in maintaining vascular homeostasis, at least partially through inhibition of EC inflammatory activation. However, the functions of endothelial KLF11 in AAA remain unknown. Here we found that endothelial KLF11 expression was reduced in the ECs from human aneurysms and was time dependently decreased in the aneurysmal endothelium from both elastase- and Pcsk9/AngII-induced AAA mouse models. KLF11 deficiency in ECs markedly aggravated AAA formation, whereas EC-selective KLF11 overexpression markedly inhibited AAA formation. Mechanistically, KLF11 not only inhibited the EC inflammatory response but also diminished MMP9 expression and activity and reduced NADPH oxidase 2-mediated production of reactive oxygen species in ECs. In addition, KLF11-deficient ECs induced smooth muscle cell dedifferentiation and apoptosis. Overall, we established endothelial KLF11 as a potentially novel factor protecting against AAA and a potential target for intervention in aortic aneurysms.
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http://dx.doi.org/10.1172/jci.insight.141673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021107PMC
March 2021

Machine Learning Applied to Determine the Molecular Descriptors Responsible for the Viscosity Behavior of Concentrated Therapeutic Antibodies.

Mol Pharm 2021 03 15;18(3):1167-1175. Epub 2021 Jan 15.

Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Predicting the solution viscosity of monoclonal antibody (mAb) drug products remains as one of the main challenges in antibody drug design, manufacturing, and delivery. In this work, the concentration-dependent solution viscosity of 27 FDA-approved mAbs was measured at pH 6.0 in 10 mM histidine-HCl. Six mAbs exhibited high viscosity (>30 cP) in solutions at 150 mg/mL mAb concentration. Combining molecular modeling and machine learning feature selection, we found that the net charge in the mAbs and the amino acid composition in the Fv region are key features which govern the viscosity behavior. For mAbs whose behavior was not dominated by charge effects, we observed that high viscosity is correlated with more hydrophilic and fewer hydrophobic residues in the Fv region. A predictive model based on the net charges of mAbs and a high viscosity index is presented as a fast screening tool for classifying low- and high-viscosity mAbs.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c01073DOI Listing
March 2021

Loss-of-function genomic variants highlight potential therapeutic targets for cardiovascular disease.

Nat Commun 2020 12 18;11(1):6417. Epub 2020 Dec 18.

The Institute for Translational Genomics and Population Sciences, Department of Pediatrics and Los Angeles Biomedical Research Institute, Harbor-UCLA, Torrance, CA, USA.

Pharmaceutical drugs targeting dyslipidemia and cardiovascular disease (CVD) may increase the risk of fatty liver disease and other metabolic disorders. To identify potential novel CVD drug targets without these adverse effects, we perform genome-wide analyses of participants in the HUNT Study in Norway (n = 69,479) to search for protein-altering variants with beneficial impact on quantitative blood traits related to cardiovascular disease, but without detrimental impact on liver function. We identify 76 (11 previously unreported) presumed causal protein-altering variants associated with one or more CVD- or liver-related blood traits. Nine of the variants are predicted to result in loss-of-function of the protein. This includes ZNF529:p.K405X, which is associated with decreased low-density-lipoprotein (LDL) cholesterol (P = 1.3 × 10) without being associated with liver enzymes or non-fasting blood glucose. Silencing of ZNF529 in human hepatoma cells results in upregulation of LDL receptor and increased LDL uptake in the cells. This suggests that inhibition of ZNF529 or its gene product should be prioritized as a novel candidate drug target for treating dyslipidemia and associated CVD.
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http://dx.doi.org/10.1038/s41467-020-20086-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7749177PMC
December 2020

Endothelial TFEB (Transcription Factor EB) Improves Glucose Tolerance via Upregulation of IRS (Insulin Receptor Substrate) 1 and IRS2.

Arterioscler Thromb Vasc Biol 2021 02 10;41(2):783-795. Epub 2020 Dec 10.

Department of Internal Medicine, Frankel Cardiovascular Center, University of Michigan Medical Center, Ann Arbor (J.S., H.L., W.L., G.Z., D.H., Z.C., Y.L., M.T.G.-B., J.Z., Y.E.C., Y.F.).

Objective: Vascular endothelial cells (ECs) play a critical role in maintaining vascular homeostasis. Aberrant EC metabolism leads to vascular dysfunction and metabolic diseases. TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, has protective effects on vascular inflammation and atherosclerosis. However, the role of endothelial TFEB in metabolism remains to be explored. In this study, we sought to investigate the role of endothelial TFEB in glucose metabolism and underlying molecular mechanisms. Approach and Results: To determine whether endothelial TFEB is critical for glucose metabolism in vivo, we utilized EC-selective TFEB knockout and EC-selective TFEB transgenic mice fed a high-fat diet. EC-selective TFEB knockout mice exhibited significantly impaired glucose tolerance compared with control mice. Consistently, EC-selective TFEB transgenic mice showed improved glucose tolerance. In primary human ECs, small interfering RNA-mediated TFEB knockdown blunts Akt (AKT serine/threonine kinase) signaling. Adenovirus-mediated overexpression of TFEB consistently activates Akt and significantly increases glucose uptake in ECs. Mechanistically, TFEB upregulates IRS1 and IRS2 (insulin receptor substrate 1 and 2). TFEB increases IRS2 transcription measured by reporter gene and chromatin immunoprecipitation assays. Furthermore, we found that TFEB increases IRS1 protein via downregulation of microRNAs (miR-335, miR-495, and miR-548o). In vivo, Akt signaling in the skeletal muscle and adipose tissue was significantly impaired in EC-selective TFEB knockout mice and consistently improved in EC-selective TFEB transgenic mice on high-fat diet.

Conclusions: Our data revealed a critical role of TFEB in endothelial metabolism and suggest that TFEB constitutes a potential molecular target for the treatment of vascular and metabolic diseases.
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http://dx.doi.org/10.1161/ATVBAHA.120.315310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105265PMC
February 2021

Human apolipoprotein A-II reduces atherosclerosis in knock-in rabbits.

Atherosclerosis 2021 01 28;316:32-40. Epub 2020 Nov 28.

Center for Advanced Models for Translational Sciences and Therapeutics, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA. Electronic address:

Background And Aims: Apolipoprotein A-II (apoAII) is the second major apolipoprotein of the high-density lipoprotein (HDL) particle, after apoAI. Unlike apoAI, the biological and physiological functions of apoAII are unclear. We aimed to gain insight into the specific roles of apoAII in lipoprotein metabolism and atherosclerosis using a novel rabbit model.

Methods: Wild-type (WT) rabbits are naturally deficient in apoAII, thus their HDL contains only apoAI. Using TALEN technology, we replaced the endogenous apoAI in rabbits through knock-in (KI) of human apoAII. The newly generated apoAII KI rabbits were used to study the specific function of apoAII, independent of apoAI.

Results: ApoAII KI rabbits expressed exclusively apoAII without apoAI, as confirmed by RT-PCR and Western blotting. On a standard diet, the KI rabbits exhibited lower plasma triglycerides (TG, 52%, p < 0.01) due to accelerated clearance of TG-rich particles and higher lipoprotein lipase activity than the WT littermates. ApoAII KI rabbits also had higher plasma HDL-C (28%, p < 0.05) and their HDL was rich in apoE, apoAIV, and apoAV. When fed a cholesterol-rich diet for 16 weeks, apoAII KI rabbits were resistant to diet-induced hypertriglyceridemia and developed significantly less aortic atherosclerosis compared to WT rabbits. HDL isolated from rabbits with apoAII KI had similar cholesterol efflux capacity and anti-inflammatory effects as HDL isolated from the WT rabbits.

Conclusions: ApoAII KI rabbits developed less atherosclerosis than WT rabbits, possibly through increased plasma HDL-C, reduced TG and atherogenic lipoproteins. These results suggest that apoAII may serve as a potential target for the treatment of atherosclerosis.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770079PMC
January 2021

Glycine-based treatment ameliorates NAFLD by modulating fatty acid oxidation, glutathione synthesis, and the gut microbiome.

Sci Transl Med 2020 12;12(572)

The Cancer Metabolism Laboratory, the Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel.

Nonalcoholic fatty liver disease (NAFLD) including nonalcoholic steatohepatitis (NASH) has reached epidemic proportions with no pharmacological therapy approved. Lower circulating glycine is consistently reported in patients with NAFLD, but the causes for reduced glycine, its role as a causative factor, and its therapeutic potential remain unclear. We performed transcriptomics in livers from humans and mice with NAFLD and found suppression of glycine biosynthetic genes, primarily alanine-glyoxylate aminotransferase 1 (). Genetic ( mice) and dietary approaches to limit glycine availability resulted in exacerbated diet-induced hyperlipidemia and steatohepatitis, with suppressed mitochondrial/peroxisomal fatty acid β-oxidation (FAO) and enhanced inflammation as the underlying pathways. We explored glycine-based compounds with dual lipid/glucose-lowering properties as potential therapies for NAFLD and identified a tripeptide (Gly-Gly-L-Leu, DT-109) that improved body composition and lowered circulating glucose, lipids, transaminases, proinflammatory cytokines, and steatohepatitis in mice with established NASH induced by a high-fat, cholesterol, and fructose diet. We applied metagenomics, transcriptomics, and metabolomics to explore the underlying mechanisms. The bacterial genus sensu stricto was markedly increased in mice with NASH and decreased after DT-109 treatment. DT-109 induced hepatic FAO pathways, lowered lipotoxicity, and stimulated de novo glutathione synthesis. In turn, inflammatory infiltration and hepatic fibrosis were attenuated via suppression of NF-κB target genes and TGFβ/SMAD signaling. Unlike its effects on the gut microbiome, DT-109 stimulated FAO and glutathione synthesis independent of NASH. In conclusion, impaired glycine metabolism may play a causative role in NAFLD. Glycine-based treatment attenuates experimental NAFLD by stimulating hepatic FAO and glutathione synthesis, thus warranting clinical evaluation.
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http://dx.doi.org/10.1126/scitranslmed.aaz2841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7982985PMC
December 2020

MiCas9 increases large size gene knock-in rates and reduces undesirable on-target and off-target indel edits.

Nat Commun 2020 11 27;11(1):6082. Epub 2020 Nov 27.

Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical School, 2800 Plymouth Road, Ann Arbor, MI, 48109, USA.

Gene editing nuclease represented by Cas9 efficiently generates DNA double strand breaks at the target locus, followed by repair through either the error-prone non-homologous end joining or the homology directed repair pathways. To improve Cas9's homology directed repair capacity, here we report the development of miCas9 by fusing a minimal motif consisting of thirty-six amino acids to spCas9. MiCas9 binds RAD51 through this fusion motif and enriches RAD51 at the target locus. In comparison to spCas9, miCas9 enhances double-stranded DNA mediated large size gene knock-in rates, systematically reduces off-target insertion and deletion events, maintains or increases single-stranded oligodeoxynucleotides mediated precise gene editing rates, and effectively reduces on-target insertion and deletion rates in knock-in applications. Furthermore, we demonstrate that this fusion motif can work as a "plug and play" module, compatible and synergistic with other Cas9 variants. MiCas9 and the minimal fusion motif may find broad applications in gene editing research and therapeutics.
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http://dx.doi.org/10.1038/s41467-020-19842-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695827PMC
November 2020

Phenotypes of CF rabbits generated by CRISPR/Cas9-mediated disruption of the CFTR gene.

JCI Insight 2021 01 11;6(1). Epub 2021 Jan 11.

Marsico Lung Institute, University of North Carolina School of Medicine, Chapel Hill, North Carolina, USA.

Existing animal models of cystic fibrosis (CF) have provided key insights into CF pathogenesis but have been limited by short lifespans, absence of key phenotypes, and/or high maintenance costs. Here, we report the CRISPR/Cas9-mediated generation of CF rabbits, a model with a relatively long lifespan and affordable maintenance and care costs. CF rabbits supplemented solely with oral osmotic laxative had a median survival of approximately 40 days and died of gastrointestinal disease, but therapeutic regimens directed toward restoring gastrointestinal transit extended median survival to approximately 80 days. Surrogate markers of exocrine pancreas disorders were found in CF rabbits with declining health. CFTR expression patterns in WT rabbit airways mimicked humans, with widespread distribution in nasal respiratory and olfactory epithelia, as well as proximal and distal lower airways. CF rabbits exhibited human CF-like abnormalities in the bioelectric properties of the nasal and tracheal epithelia. No spontaneous respiratory disease was detected in young CF rabbits. However, abnormal phenotypes were observed in surviving 1-year-old CF rabbits as compared with WT littermates, and these were especially evident in the nasal respiratory and olfactory epithelium. The CF rabbit model may serve as a useful tool for understanding gut and lung CF pathogenesis and for the practical development of CF therapeutics.
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http://dx.doi.org/10.1172/jci.insight.139813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821608PMC
January 2021

Synthetic high-density lipoproteins delivering liver X receptor agonist prevent atherogenesis by enhancing reverse cholesterol transport.

J Control Release 2021 01 11;329:361-371. Epub 2020 Nov 11.

Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, MI 48109, United States; Biointerfaces Institute, University of Michigan, Ann Arbor, MI 48109, United States. Electronic address:

Liver X nuclear receptor (LXR) agonists are promising anti-atherosclerotic agents that increase the expression of cholesterol transporters on atheroma macrophages leading to increased efflux of cholesterol to endogenous high-density lipoprotein (HDL) acceptors. HDL subsequently delivers effluxed cholesterol to the liver by the process of reverse cholesterol transport, resulting in reduction of atherosclerotic plaques. However, LXR agonists administration triggers undesirable liver steatosis and hypertriglyceridemia due to increased fatty acid and sterol synthesis. LXR-induced liver toxicity, poor drug aqueous solubility and low levels of endogenous HDL acceptors in target patient populations limit the clinical translation of LXR agonists. Here, we propose a dual-antiatherogenic strategy for administration of the LXR agonist, T0901317 (T1317), by encapsulating in synthetic HDL (sHDL) nanoparticles. sHDL had been clinically proven to serve as cholesterol acceptors, resulting in plaque reduction in atherosclerosis patients. In addition, the hydrophobic core and endogenous atheroma-targeting ability of sHDL allow for encapsulation of water-insoluble drugs and their subsequent delivery to atheroma. Several compositions of sHDL were tested to optimize both T1317 encapsulation efficiency and ability of T1317-sHDL to efflux cholesterol. Optimized T1317-sHDL exhibited more efficient cholesterol efflux from macrophages and enhanced atheroma-targeting relative to free drug. Most importantly, in an apolipoprotein E deficient (ApoE) atherosclerosis progression murine model, T1317-sHDL showed superior inhibition of atherogenesis and reduced hypertriglyceridemia side effects in comparison to the free drug and blank sHDL. The T1317-sHDL pharmacological efficacy was observed at doses lower than those previously described for LXR agents, which may have additional safety benefits. In addition, the established clinical manufacturing, safety and efficacy of blank sHDL nanoparticles used in this study could facilitate future clinical translation of LXR-loaded sHDLs.
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http://dx.doi.org/10.1016/j.jconrel.2020.11.016DOI Listing
January 2021

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications.

CNS Neurol Disord Drug Targets 2020 Oct 26. Epub 2020 Oct 26.

Department of Anatomy, Neuroscience Laboratory for Cognitive and Developmental Disorders, Medical College of Jinan University, Guangzhou, 510630. China.

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury.

Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated.

Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin.

Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites.

Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules.

Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.
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http://dx.doi.org/10.2174/1871527319666201026165855DOI Listing
October 2020
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