Specific Deletion Research Articles

Pannexin1 Mediates Early-Life Seizure-Induced Social Behavior Deficits.

There is a high co-morbidity between childhood epilepsy and autism spectrum disorder (ASD), with age of seizure onset being a critical determinant of behavioral outcomes. The interplay between these comorbidities has been investigated in animal models with results showing that the induction of seizures at early post-natal ages leads to learning and memory deficits and to autistic-like behavior in adulthood. Modifications of the excitation/inhibition (glutamate/GABA, ATP/adenosine) balance that follows early-life seizures (ELS) are thought to be the physiological events that underlie neuropsychiatric and neurodevelopmental disorders. Although alterations in purinergic/adenosinergic signaling have been implicated in seizures and ASD, it is unknown whether the ATP release channels, Pannexin1 (Panx1), contribute to ELS-induced behavior changes. To tackle this question, we used the ELS-kainic acid model in transgenic mice with global and cell type specific deletion of Panx1 to evaluate whether these channels were involved in behavioral deficits that occur later in life. Our studies show that ELS results in Panx1 dependent social behavior deficits and also in poor performance in a spatial memory test that does not involve Panx1. These findings provide support for a link between ELS and adult behavioral deficits. Moreover, we identify neuronal and not astrocyte Panx1 as a potential target to specifically limit astrogliosis and social behavioral deficits resultant from early-life seizures.

IL-10 dampens antitumor immunity and promotes liver metastasis via PD-L1 induction

The liver is one of the organs most commonly affected by metastasis. The presence of liver metastases has been reported to be responsible for an immunosuppressive microenvironment and diminished immunotherapy efficacy. Herein, we aimed to investigate the role of IL-10 in liver metastasis and to determine how its modulation could affect the efficacy of immunotherapy invivo. To induce spontaneous or forced liver metastasis in mice, murine cancer cells (MC38) or colon tumor organoids were injected into the cecum or the spleen, respectively. Mice with complete and cell type-specific deletion of IL-10 and IL-10 receptor alpha were used to identify the source and the target of IL-10 during metastasis formation. Programmed death ligand 1 (PD-L1)-deficient mice were used to test the role of this checkpoint. Flow cytometry was applied to characterize the regulation of PD-L1 by IL-10. We found that Il10-deficient mice and mice treated with IL-10 receptor alpha antibodies were protected against liver metastasis formation. Furthermore, by using IL-10 reporter mice, we demonstrated that Foxp3+ regulatory T cells (Tregs) were the major cellular source of IL-10 in liver metastatic sites. Accordingly, deletion of IL-10 in Tregs, but not in myeloid cells, led to reduced liver metastasis. Mechanistically, IL-10 acted on Tregs in an autocrine manner, thereby further amplifying IL-10 production. Furthermore, IL-10 acted on myeloid cells, i.e. monocytes, and induced the upregulation of the immune checkpoint protein PD-L1. Finally, the PD-L1/PD-1 axis attenuated CD8-dependent cytotoxicity against metastatic lesions. Treg-derived IL-10 upregulates PD-L1 expression in monocytes, which in turn reduces CD8+ T-cell infiltration and related antitumor immunity in the context of colorectal cancer-derived liver metastases. These findings provide the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastases. Liver metastasis diminishes the effectiveness of immunotherapy and increases the mortality rate in patients with colorectal cancer. We investigated the role of IL-10 in liver metastasis formation and assessed its impact on the effectiveness of immunotherapy. Our data show that IL-10 is a pro-metastatic factor involved in liver metastasis formation and that it acts as a regulator of PD-L1. This provides the basis for future monitoring and targeting of IL-10 in colorectal cancer-derived liver metastasis.

Macrophage-specific deletion of Notch-1 induced M2 anti-inflammatory effect in atherosclerosis via activation of the PI3K-oxidative stress axis.

Notch-1 signaling is significantly associated with the occurrence and development of atherosclerosis (AS). However, the molecular mechanisms underlying the specific deletion of Notch-1 in AS-associated macrophages are not fully understood. This study aimed to investigate the effects of Notch-1 in AS. Tissue samples were obtained from atherosclerotic segments of human carotid arteries. Immunofluorescence staining showed that Notch-1 was significantly colocalized with macrophages (CD68+), and Notch-1 staining was increased in human vulnerable plaques. Notch-1MAC-KO/ApoE-/- mice were generated in which Notch-1 was selectively inactivated in macrophages, and WT for littermate control mice (ApoE-/-/Notch-1WT). A control group was then established. All mice fed with a high-fat and Oil Red O, Movat, a-SMA, CD68, and Sirius red staining were used to evaluate the morphology. Specific deletion of Notch-1 in macrophages repressed the pathophysiology of AS. Immunofluorescent staining and Western blotting revealed that Notch-1MAC-KO repressed M1 and M2 responses in AS. Here, GSEA revealed that Notch-1 activation and PI3K signaling were statistically significantly correlated with each other, and Notch-1 was involved in the regulation of the PI3K signaling pathway. In the in vitro experiments, the secretion of Arg-1 and exosomes was classified by peritoneal macrophages of Notch-1MAC-KO/ApoE-/- and Notch-1WT/ApoE-/- mice. Immunohistochemistry staining and Western blotting were used to measure the expression levels of Notch1, PI3K, p-PI3K, AKT, p-AKT, Arg-1, IL-6, CD36, SREBP-1, CD206, iNOS, cleaved-caspase-3/-9, Bax, CD9, Alix and TSG101 in the peritoneal macrophages and exosomes, respectively. The specific deletion of Notch-1 in macrophage represses the formation and development of AS via the PI3K/AKT signaling pathway.

Alcohol induced behavioral and immune perturbations are attenuated by activation of CB2 cannabinoid receptors.

The endocannabinoidome (eCBome) is the expanded endocannabinoid system (ECS) and studies show that there is a link between this system and how it modulates alcohol induced neuroinflammation. Using conditional knockout (cKO) mice with selective deletion of cannabinoid type 2 receptors (CB2Rs) in dopamine neurons (DAT-Cnr2) and in microglia (Cx3Cr1-Cnr2), we investigated how CB2Rs modulate behavioral and neuroinflammation induced by alcohol. Behavioral tests including locomotor and wheel running activity, rotarod performance test, and alcohol preference tests were used to evaluate behavioral changes induced by alcohol. Using ELISA assay, we investigated the level of pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1α (IL-1α), and interleukin-1β (IL-1β) in the hippocampus of mice. The findings demonstrated that locomotor activity, wheel running, and rotarod performance activities were significantly affected by cell-type specific deletion of CB2Rs in dopamine neurons and microglia. The non-selective CB2R agonist, WIN 55,212-2, reduced alcohol preference in the wild type and cell-type specific CB2R cKO mice. In addition, the result showed that cell-type specific deletion of CB2Rs per se and administration of alcohol to CB2R cKO mice increased the expression of proinflammatory cytokines in the hippocampus. These findings suggest the involvement of CB2Rs in modulating behavioral and immune alterations induced by alcohol.

De novel heterozygous copy number deletion on 7q31.31-7q31.32 involving TSPAN12 gene with familial exudative vitreoretinopathy in a Chinese family.

To investigate the genetic and clinical characteristics of patients with a large heterozygous copy number deletion on 7q31.31-7q31.32. A family with familial exudative vitreoretinopathy (FEVR) phenotype was included in the study. Whole-exome sequencing (WES) was initially used to locate copy number variations (CNVs) on 7q31.31-31.32, but failed to detect the precise breakpoint. The long-read sequencing, Oxford Nanopore sequencing Technology (ONT) was used to get the accurate breakpoint which is verified by quantitative real-time polymerase chain reaction (QPCR) and Sanger Sequencing. The proband, along with her father and younger brother, were found to have a heterozygous 4.5 Mb CNV deletion located on 7q31.31-31.32, which included the FEVR-related gene TSPAN12. The specific deletion was confirmed as del(7)(q31.31q31.32)chr7:g.119451239_123956818del. The proband exhibited a phase 2A FEVR phenotype, characterized by a falciform retinal fold, macular dragging, and peripheral neovascularization with leaking of fluorescence. These symptoms led to a significant decrease in visual acuity in both eyes. On the other hand, the affected father and younger brother showed a milder phenotype. The heterozygous CNV deletion located on 7q31.31-7q31.32 is associated with the FEVR phenotype. The use of long-read sequencing techniques is essential for accurate molecular diagnosis of genetic disorders.

Myeloid deletion of talin-1 reduces mucosal macrophages and protects mice from colonic inflammation

The intestinal immune response is crucial in maintaining a healthy gut, but the enhanced migration of macrophages in response to pathogens is a major contributor to disease pathogenesis. Integrins are ubiquitously expressed cellular receptors that are highly involved in immune cell adhesion to endothelial cells while in the circulation and help facilitate extravasation into tissues. Here we show that specific deletion of the Tln1 gene encoding the protein talin-1, an integrin-activating scaffold protein, from cells of the myeloid lineage using the Lyz2-cre driver mouse reduces epithelial damage, attenuates colitis, downregulates the expression of macrophage markers, decreases the number of differentiated colonic mucosal macrophages, and diminishes the presence of CD68-positive cells in the colonic mucosa of mice infected with the enteric pathogen Citrobacter rodentium. Bone marrow-derived macrophages lacking expression of Tln1 did not exhibit a cell-autonomous phenotype; there was no impaired proinflammatory gene expression, nitric oxide production, phagocytic ability, or surface expression of CD11b, CD86, or major histocompatibility complex II in response to C. rodentium. Thus, we demonstrate that talin-1 plays a role in the manifestation of infectious colitis by increasing mucosal macrophages, with an effect that is independent of macrophage activation.

The tubulin database: Linking mutations, modifications, ligands and local interactions.

Microtubules are polymeric filaments, constructed of α-β tubulin heterodimers that underlie critical subcellular structures in eukaryotic organisms. Four homologous proteins (γ-, δ-, ε- and ζ-tubulin) additionally contribute to specialized microtubule functions. Although there is an immense volume of publicly available data pertaining to tubulins, it is difficult to assimilate all potentially relevant information across diverse organisms, isotypes, and categories of data. We previously assembled an extensive web-based catalogue of published missense mutations to tubulins with >1,500 entries that each document a specific substitution to a discrete tubulin, the species where the mutation was described and the associated phenotype with hyperlinks to the amino acid sequence and citation(s) for research. This report describes a significant update and expansion of our online resource (TubulinDB.bio.uci.edu) to nearly 18,000 entries. It now encompasses a cross-referenced catalog of post-translational modifications (PTMs) to tubulin drawn from public datasets, primary literature, and predictive algorithms. In addition, tubulin protein structures were used to define local interactions with bound ligands (GTP, GDP and diverse microtubule-targeting agents) and amino acids at the intradimer interface, within the microtubule lattice and with associated proteins. To effectively cross-reference these datasets, we established a universal tubulin numbering system to map entries into a common framework that accommodates specific insertions and deletions to tubulins. Indexing and cross-referencing permitted us to discern previously unappreciated patterns. We describe previously unlinked observations of loss of PTM sites in the context of cancer cells and tubulinopathies. Similarly, we expanded the set of clinical substitutions that may compromise MAP or microtubule-motor interactions by collecting tubulin missense mutations that alter amino acids at the interface with dynein and doublecortin. By expanding the database as a curated resource, we hope to relate model organism data to clinical findings of pathogenic tubulin variants. Ultimately, we aim to aid researchers in hypothesis generation and design of studies to dissect tubulin function.

Menin Reduces Parvalbumin Expression and is Required for the Anti-Depressant Function of Ketamine.

Dysfunction of parvalbumin (PV) neurons is closely involved in depression, however, the detailed mechanism remains unclear. Based on the previous finding that multiple endocrine neoplasia type 1 (Protein: Menin; Gene: Men1) mutation (G503D) is associated with a higher risk of depression, a Menin-G503D mouse model is generated that exhibits heritable depressive-like phenotypes and increases PV expression in brain. This study generates and screens a serial of neuronal specific Men1 deletion mice, and found that PV interneuron Men1 deletion mice (PcKO) exhibit increased cortical PV levels and depressive-like behaviors. Restoration of Menin, knockdown PV expression or inhibition of PV neuronal activity in PV neurons all can ameliorate the depressive-like behaviors of PcKO mice. This study next found that ketamine stabilizes Menin by inhibiting protein kinase A (PKA) activity, which mediates the anti-depressant function of ketamine. These results demonstrate a critical role for Menin in depression, and prove that Menin is key to the antidepressant function of ketamine.

Microglial deletion of C1q rescues AD cognitive decline and synaptic loss

AbstractBackgroundThe complement (C’) system contributes to enhanced inflammation and cognitive decline in Alzheimer’s disease (AD). Previous studies have demonstrated constitutive deletion of the classical initiator protein, C1q, reduces pathology and synaptic loss in AD mouse model. As it is now known that microglia are the primary producers of C1q in the brain, the main objective of this study was to determine if microglial specific deletion of C1q would rescue cognitive impairment and synaptic loss in AD.MethodC1qaFLlFL:CX3CR1Cre (designated C1qΔMG)mice, which experience tamoxifen‐independent deletion of microglial C1q by 2 months of age, were crossed to Arctic: C1qaFl/Fl: mice to generate WT and Artic mice with and without the CX3CR1Cre transgene. At 10 months of age, mice underwent behavioral testing of hippocampal dependent spatial memory, including object location memory (OLM) and spatial reference Y‐maze. Tissue was collected at the end of testing and immunostained for pre (VGlut1) and post (PSD95) synaptic makers and Z‐stacks using superresolution microscopy in the CA1 of the hippocampus were obtained. The density of pre‐ and post‐synaptic puncta and their colocalization was assessed using IMARIS.ResultWhile both male and female Arctic mice displayed a significant impairment in long‐term memory, as assessed by OLM, microglial deletion of C1q rescued cognitive deficits in male but not female Arctic mice. Male Arctic C1qΔMGmice also performed better on Y‐maze testing than Arctic controls. Similar sex‐dependent results were observed in synaptic density. Female Arctic C1qΔMG mice displayed reduced levels of PSD95 and Vglut1 puncta compared to female Arctic mice, as well as a trending reduction in colocalization. In contrast, male Arctic C1qΔMG mice displayed increased levels of PSD95, trending reductions in VGlut1 compared to male Arctic controls, and trending increases in synaptic colocalization, suggesting an increase in functional synapses in the male mouse.ConclusionDeletion of C1q in microglia prior to amyloid plaque deposition protects against cognitive decline and synaptic loss in a greater extent in males than females.

Blimp-1 and c-Maf regulate Il10 andnegatively regulate common and unique proinflammatory gene networks in IL-12 plus IL-27-driven T helper-1 cells.

CD4 + Th1 cells producing IFN-γ are required to eradicate intracellular pathogens, however if uncontrolled these cells can cause immunopathology. The cytokine IL-10 is produced by multiple immune cells including Th1 cells during infection and regulates the immune response to minimise collateral host damage. In this study we aimed to elucidate the transcriptional network of genes controlling the expression of Il10 and proinflammatory cytokines, including Ifng in Th1 cells differentiated from mouse naive CD4 + T cells. We applied computational analysis of gene regulation derived from temporal profiling of gene expression clusters obtained from bulk RNA sequencing (RNA-seq) of flow cytometry sorted naïve CD4 + T cells from mouse spleens differentiated in vitro into Th1 effector cells with IL-12 and IL-27 to produce Ifng and Il10, compared to IL-27 alone which express Il10 only , or IL-12 alone which express Ifng and no Il10, or medium control driven-CD4 + T cells which do not express effector cytokines . Data were integrated with analysis of active genomic regions from these T cells using an assay for transposase-accessible chromatin with sequencing (ATAC)-seq, integrated with literature derived-Chromatin-immunoprecipitation (ChIP)-seq data and the RNA-seq data, to elucidate the transcriptional network of genes controlling expression of Il10 and pro-inflammatory effector genes in Th1 cells. The co-dominant role for the transcription factors, Prdm1 (encoding Blimp-1) and Maf (encoding c-Maf) , in cytokine gene regulation in Th1 cells, was confirmed using T cells obtained from mice with T-cell specific deletion of these transcription factors. We show that the transcription factors Blimp-1 and c-Maf each have unique and common effects on cytokine gene regulation and not only co-operate to induce Il10 gene expression in IL-12 plus IL-27 differentiated mouse Th1 cells, but additionally directly negatively regulate key proinflammatory cytokines including Ifng, thus providing mechanisms for reinforcement of regulated Th1 cell responses. These data show that Blimp-1 and c-Maf positively and negatively regulate a network of both unique and common anti-inflammatory and pro-inflammatory genes to reinforce a Th1 response in mice that will eradicate pathogens with minimum immunopathology.

A Gad2 specific Slc6a8 deletion recapitulates the contextual and cued freezing deficits seen in Slc6a8-/y mice

The creatine (Cr)-phosphocreatine shuttle is essential for ATP homeostasis. In humans, the absence of brain Cr causes significant intellectual disability, epilepsy, and language delay. Mutations of the creatine transporter (SLC6A8) are the most common cause of Cr deficiency. In rodents, Slc6a8 deletion causes deficits in spatial learning, novel object recognition (NOR), as well as in contextual and cued freezing. The mechanisms that underlie these cognitive deficits are not known. Due to the heterogeneous nature of the brain, it is important to determine which systems are affected by a loss of Cr. In this study, we generated mice lacking Slc6a8 in GABAergic neurons by crossing Slc6a8FL mice with Gad2-Cre mice. These Gad2-specific Slc6a8 knockout (cKO) mice, along with the ubiquitous Slc6a8 KO (Slc6a8-/y), Gad2-Cre+, and wild-type (WT) mice were tested in the Morris water maze, NOR, conditioned freezing, and the radial water maze. Similar to the Slc6a8-/y mice, cKO mice had reduced contextual and cued freezing compared with WT mice. The cKO mice had a mild spatial learning deficit during the reversal phase of the MWM, however they were not as pronounced as in Slc6a8-/y mice. In NOR, the Gad2-Cre mice spent less time with the novel object, similar to the reduced novel time in the cKO mice. There were no changes in radial water maze performance. Slc6a8 deletion in GABAergic neurons is sufficient to recapitulate the conditioned freezing deficits seen in Slc6a8-/y mice.

PD-1 Expression By Dendritic Cells Is a Key Regulator of T-Cell Immunity in Cancer

Programmed cell death 1 (PD-1) is an inhibitory receptor expressed on a variety of immune cells and a therapeutic target of checkpoint immunotherapy for cancer patients. PD-1 engagement by PD-L1 or PD-L2 can inhibit activation and expansion of tumor antigen-specific T cells. PD-1 expression in tumor-associated macrophages (TAM) correlates with impaired phagocytosis and antigen presenting function and enhanced immunosuppression. PD-1 is also expressed in dendritic cells (DC) and has been shown to correlate negatively with the outcome of DC-based cancer vaccines. On DC, PD-1 can interact with PD-L1 in cis when both of these receptors are expressed on the same cell surface. DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, and PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Very little is known about the specific role of PD-1 on DCs function. In the present study, we sought to understand the role of PD-1 on DCs in the context of cancer. We generated mice with conditional targeting of the Pdcd1 gene (encoding for PD-1) and crossed them with CD11cCre ( Pdcd1 f/fCD11cCre) or Clec9Cre ( Pdcd1 f/fClec9Cre) resulting in specific deletion of PD-1 in DC cells. We used the MC17 fibrosarcoma syngeneic mouse model to investigate the tentative implications of DC-specific PD-1 ablation on anti-tumor responses. We found larger tumor sizes and weights in tumor-bearing Pdcd1 f/fCD11cCre mice compared to their Pdcd1 wt/wtCD11cCre control counterparts. Pdcd1 f/fCD11cCre tumor-bearing mice had significantly higher fractions of DCs in spleens and tumor-draining lymph nodes (tdLNs) but only minimal differences in their activation state and checkpoint marker expression. Similarly, CD4 + and CD8 + T cells from spleens and tdLNs were comparable in percentages and activation states. However, characterization of the immune cells at the tumor site, indicated that Pdcd1 f/fCD11cCre tumor-bearing mice had a more immunosuppressive tumor microenvironment with increased fractions of CD11b +Ly6C hiLy6G -monocytic myeloid derived suppressor cells (M-MDSCs) compared to control tumor-bearing mice. Moreover, flow cytometric analysis revealed that intratumoral M-MDSC in Pdcd1 f/fCD11cCre mice displayed enhanced expression of PD-L1 and CD38, consistent with a more activated and more immunosuppressive phenotype. Characterization of tumor infiltrating CD4 + and CD8 + T cells also revealed vast differences in composition and activation states. Specifically, Pdcd1 f/fCD11cCre mice had larger populations of intratumoral CD3 + T cells, with a greater CD4/CD8 T cell ratio, and higher percentages of CD4 + T regulatory cells. Conversely, intratumoral CD8 + T cells were reduced, and were characterized by diminished expansion of CD8 + T effector cells, and a less activated phenotype as determined by lower expression of CD25, CD69, and GITR. In syngeneic tumor experiments with B16 melanoma expressing ovalbumin (B16-Ova), Pdcd1 f/fCD11cCre mice and Pdcd1 f/fClec9Cre mice similarly had greater tumor sizes and weights compared to their control counterparts. These results indicate that selective ablation of PD-1 in DCs cells confers a more immunosuppressive tumor microenvironment by promoting T regulatory cell expansion and diminishing CD8 + T effector cell activation compromising anti-tumor responses.

Fatty acids and inflammatory stimuli induce expression of long-chain acyl-CoA synthetase 1 to promote lipid remodeling in diabetic kidney disease

Fatty acid handling and complex lipid synthesis are altered in the kidney cortex of diabetic patients. We recently showed that inhibition of the renin-angiotensin system without changes in glycemia can reverse diabetic kidney disease (DKD) and restore the lipid metabolic network in the kidney cortex of diabetic (db/db) mice, raising the possibility that lipid remodeling may play a central role in DKD. However, the roles of specific enzymes involved in lipid remodeling in DKD have not been elucidated. In the present study, we used this diabetic mouse model and a proximal tubule epithelial cell line (HK2) to investigate the potential relationship between long-chain acyl-CoA synthetase 1 (ACSL1) and lipid metabolism in response to fatty acid exposure and inflammatory signals. We found ACSL1 expression was significantly increased in the kidney cortex of db/db mice, and exposure to palmitate (PA) or tumor necrosis factor (TNF-α) significantly increased Acsl1 mRNA expression in HK-2 cells. In addition, PA treatment significantly increased the levels of long-chain acylcarnitines and fatty acyl CoAs in HK2 cells, and these increases were abolished in HK2 cell lines with specific deletion of Acsl1(Acsl1KO cells), suggesting a key role for ACSL1 in fatty acid β-oxidation. In contrast, TNF-α treatment significantly increased the levels short-chain acylcarnitines and long-chain fatty acyl CoAs in HK2 cells but not in Acsl1KO cells, consistent with fatty acid channeling to complex lipids. Taken together, our data demonstrate a key role for ACSL1 in regulating lipid metabolism, fatty acid partitioning, and inflammation.

Von Willebrand factor promotes radiation-induced intestinal injury (RIII) development and its cleavage enzyme rhADAMTS13 protects against RIII by reducing inflammation and oxidative stress

Patients with abdominopelvic cancer undergoing radiotherapy commonly develop radiation-induced intestinal injury (RIII); however, its underlying pathogenesis remains elusive. The von Willebrand factor (vWF)/a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in thrombosis, inflammation, and oxidative stress. However, its role in RIII remains unclear. In this study, the effect of radiation on vWF and ADAMTS13 expression was firstly evaluated in patients with cervical cancer undergoing radiotherapy and C57BL/6J mice exposed to different doses of total abdominal irradiation. Then, mice with the specific deletion of vWF in the platelets and endothelium were established to demonstrate the contribution of vWF to RIII. Additionally, the radioprotective effect of recombinant human (rh) ADAMTS13 against RIII was assessed. Results showed that both the patients with cervical cancer undergoing radiotherapy and RIII mouse model exhibited increased vWF levels and decreased ADAMTS13 levels. The knockout of platelet- and endothelium-derived vWF rectified the vWF/ADAMTS13 axis imbalance; improved intestinal structural damage; increased crypt epithelial cell proliferation; and reduced radiation-induced apoptosis, inflammation, and oxidative stress, thereby alleviating RIII. Administration of rhADAMTS13 could equally alleviate RIII. Our results demonstrated that abdominal irradiation affected the balance of the vWF/ADAMTS13 axis. vWF exerted a deleterious role and ADAMTS13 exhibited a protective role in RIII progression. rhADAMTS13 has the potential to be developed into a radioprotective agent.

Effects of N-glycan modifications on spike expression, virus infectivity, and neutralization sensitivity in ancestral compared to Omicron SARS-CoV-2 variants.

The SARS-CoV-2 spike glycoprotein has 22 potential N-linked glycosylation sites per monomer that are highly conserved among diverse variants, but how individual glycans affect virus entry and neutralization of Omicron variants has not been extensively characterized. Here we compared the effects of specific glycan deletions or modifications in the Omicron BA.1 and D614G spikes on spike expression, processing, and incorporation into pseudoviruses, as well as on virus infectivity and neutralization by therapeutic antibodies. We found that loss of potential glycans at spike residues N717 and N801 each conferred a loss of pseudovirus infectivity for Omicron but not for D614G or Delta variants. This decrease in infectivity correlated with decreased spike processing and incorporation into Omicron pseudoviruses. Oligomannose-enriched Omicron pseudoviruses generated in GnTI- cells or in the presence of kifunensine were non-infectious, whereas D614G or Delta pseudoviruses generated under similar conditions remained infectious. Similarly, growth of live (authentic) SARS-CoV-2 in the presence of kifunensine resulted in a greater reduction of titers for the BA.1.1 variant than Delta or D614G variants relative to their respective, untreated controls. Finally, we found that loss of some N-glycans, including N343 and N234, increased the maximum percent neutralization by the class 3 S309 monoclonal antibody against D614G but not BA.1 variants, while these glycan deletions altered the neutralization potency of the class 1 COV2-2196 and Etesevimab monoclonal antibodies without affecting maximum percent neutralization. The maximum neutralization by some antibodies also varied with the glycan composition, with oligomannose-enriched pseudoviruses conferring the highest percent neutralization. These results highlight differences in the interactions between glycans and residues among SARS-CoV-2 variants that can affect spike expression, virus infectivity, and susceptibility of variants to antibody neutralization.

Conditional knock out of cytosolic thioredoxin (Trx) in cardiomyocytes impairs cardiac function and exacerbates ischemia-reperfusion injury

Abstract Introduction Coronary heart disease with myocardial infarction (MI) is a leading cause of mortality in the world despite timely reperfusion of occluded coronary artery. Myocardial damage due to restoration of blood flow, known as reperfusion injury account for about 50% of final MI size. The role of Trx in protection against myocardial ischemia reperfusion has been reported; however, these studies were mostly performed using ex vivo isolated mouse/rat heart with retrograde perfusion or with injected rhTrx. However, the role of loss of Trx specifically in the myocytes using a conditional deletion has never been reported. Here, we determined whether loss of Trx in myocytes would promote ischemia-reperfusion injury, even if the endocardial endothelial cells express normal levels of Trx. Purpose We investigated the effect of specific deletion of Trx in cardiomyocytes on base-line cardiac function and cardiac function in ischemia-reperfusion of heart using conditional knockout of Trx (aMHC-Trx-KO) in the cardiomyocytes with normal levels of Trx in the endothelial cells to determine specifc contribution of Trx in cardiac function in I/R. Methods A Trx floxed mouse line was created with introduction of loxP sites flanking the mouse Trx gene. Further, the conditional deletion of Trx in cardiomyocytes was produced by breeding homozygous Trx-floxed (Trxfl/fl) mice with aMHC-Cre mice strain. The resulting aMHC-Trx-KO mice was identified by genotyping and conditional deletion of Trx in myocytes was achieved by injecting Tomoxifen (75mg/Kg in oil). The aMHC-Trx-KO mice were subjected to 30 minutes of ischemia by ligation of left descending coronary artery(LAD) and reperfusion was performed by release of slipknot and allowing blood flow of 60 minutes before sacrificing animals. The animal protocol was approved by the IACUC of the Texas Tech University Health Sciences Center, Lubbock. Echocardiography on animals was performed using Vevo 3100 (Visual Sonic). Statistical analysis was performed by Prism 8.0 (GraphPad). Results Deletion of Trx caused significant cardiac dysfunction in aMHC-Trx-KO mice compared to Trxfl/fl mice without treatment with tomoxifen. The ejection fraction (EF%, 52.82 vs. 73.83) and fractional shortening (FS%, 41.42 vs.25.89) were significantly reduced. Further, these mice subjected to I/R showed exacerbated cardiac dysfunction with significant decrease in heart rate and did not survive the reperfusion. We also found significant mitochondrial structural abnormalities and mitochondrial dysfunction in aMHC-Trx-KO mice compared to non-transgenic or Trxfl/fl mice without tomoxifen treatment. Conclusion Deletion of Trx in cardiomyocyte causes significant myocardial dysfunction. Further, Trx is required to protect against myocardial dysfunction in ischemia-reperfusion injury. Our data also suggest that cardiac mitochondria is specifically impacted by loss of Trx deletion.Figure-1

Abstract 15537: The Role of Liver Epsins in Nonalcoholic Steatohepatitis

Introduction: Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that is associated with high risks of cardiovascular diseases. Epsin is an evolutionarily conserved protein family that is involved in clathrin-mediated endocytosis. We found that both Epsin1/2 expressions in the liver are significantly up-regulated by the Western Diet (WD) induced NASH mice model, as well as in the liver biopsy of NASH patients. Hypothesis: We hypothesize that the expressions of Epsin1 and Epsin2 in the liver are required for NASH progression. Methods: We conditionally deleted Epsin1/2 using Albumin-Cre (Liver-DKO). We then induced a mouse model of NASH by feeding both WT and Liver-DKO mice a WD for 52 weeks. We used single cell multiome, combined with molecular approaches to study the mechanisms. We tested a strategy to deliver Epsin1/2 siRNAs encapsulated nanoparticles (NPs) targeting the liver via intravenous injection to treat diet induced NASH in mice. Results: We found WD-fed Liver-DKO mice have significant reductions in hepatic fibrosis formation and less lipid accumulation, and this was associated with better glucose and insulin tolerance, as well as lower serum aspartate transaminase (AST) and alanine aminotransferase (ALT) compared with WD-fed WT mice. Through analysis of the single cell multiome from the WT and Liver-DKO livers, we found more Kupffer cell in WT NASH mice compared with DKO. Interestingly, monocyte-derived inflammantory Kupffer cell (KCs) are enriched in WD-fed WT mice, however, embryonic-derived KCs are the enriched in liver DKO, and associate with dramatically reduced total KCs population. We also found that HNF4a was dramatically reduced in WD-fed WT mice, but its expression was preserved in WD-fed Liver-DKO mice, suggesting the loss of hepatocyte identity was inhibited by Epsins deletion. Therapeutically, we found significant resolution of hepatic fibrosis after knockdown of Epsin1/2 siRNA NP treatment. Conclusions: Liver specific deletion of Epsin1/2 significantly inhibits NASH progression by suppression of inflammation that protects hepatocytes from severe chronic injury. Our preclinical data suggests specially targeting Epsin1 and Epsin2 in the liver could be a promising therapeutic strategy for NASH treatment.

Abstract 14818: Endothelial Specific Deletion of Uchl1 Attenuates the Development of Pulmonary Hypertension

Introduction/Background: While ubiquitination of proteins plays an important role in the development of pulmonary arterial hypertension (PAH), little is known about the role of deubiquitinases (DUBs) and, more specifically, ubiquitin carboxyl-terminal esterase-L1 (UCHL1), in this process. Our prior work reported that reductions in endothelial UCHL1 lead to increased degradation and reduced activated levels of Akt1. Given the pathologic role of Akt1 in PAH, we now hypothesize that endothelial UCHL1 deficiency leads to reductions in activated Akt1, resulting in the attenuation of PAH. Methods/Results: Tissues from animal PH models, as well as human PA endothelial cells (hPAEC) from patients with PAH, exhibited increased vascular staining and protein expression of UCHL1. Inhibition of UCHL1 in cell culture reduced vascular endothelial growth factor (VEGF)-induced angiogenesis, proliferation and migration of hPAECs. Endothelial-specific Uchl1 knockout mice ( Uchl1 EC-/- ) demonstrated reduced right ventricular hypertrophy (RVH), RV systolic pressures (RVSP), and improved right heart function compared to control mice exposed to chronic hypoxia. Consistent with these findings, histologic examination revealed significantly improved vascular medial thickness and remodeling as well as reduced total and activated Akt1 levels in the lungs of hypoxia-exposed Uchl1 EC-/- mice compared to hypoxia-exposed control mice. Conclusions: Our findings reveal that a specific deficiency of Uchl1 in the endothelium results in PAH attenuation via reduced Akt1, highlighting a novel therapeutic target.

Abstract 15266: Inhibiting Endothelial Mammalian Sterile 20-Like Kinase 1 Attenuates Acute Lung Injury in Mice

Introduction: Lung endothelium plays a pivotal role in the orchestration of inflammatory and injury responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1), a mammalian homolog of Hippo, is a serine/threonine kinase that is ubiquitously expressed in many tissues and has been shown to play an important role in the regulation of complex biological processes, such as apoptosis, oncogenesis and organ growth. While Mst1 is highly expressed in the lung, its role in the endothelial response to pulmonary insults is largely unknown. Hypothesis: Mst1 regulates inflammatory responses in acute lung injury. Methods: Mst1 activity was assessed in lung endothelium by western blot and immunofluorescent staining. EC activation was measured by nuclear factor kappa B (NF-κB) dependent luciferase assay and the electrophoresis mobility shift assay. Mst1 knockout mice and pharmacological inhibitor were employed to assess the effects of Mst1 on homeostatic and lipopolysaccharide (LPS)-induced endothelial inflammation. Results: In this study, we found that Mst1 activation, as determined by Mst1 phosphorylation and nuclear translocation, was significantly increased in lung ECs after exposure to tumor necrosis factor-alpha (TNF-α) and in lungs of LPS-instilled mice as compared to the vehicle control. Overexpression of either full length Mst1 or its kinase domain promoted NF-κB activation by approximately 6.5-fold and 8-fold, respectively, whereas overexpression of the dominant negative forms of Mst1 attenuated endothelial inflammatory responses to both TNF-α and interleukin-1β stimulation. Consistent with these observations, endothelial specific deletion of Mst1 markedly attenuated LPS-induced acute lung injury in mice. Furthermore, we found that treatment of wild-type mice with Mst1 specific pharmacological inhibitor significantly prevented LPS-induced lung injury. Conclusion: Our findings identified Mst1 kinase as a key regulator in promoting lung EC activation and vascular inflammation, and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective for preventing inflammatory vascular diseases of the lung.

Abstract 12901: ADAR1 RNA Editing in Vascular Smooth Muscle Maintains Vascular Integrity, Regulates Cell State Transition, and is a Novel Mechanism of Coronary Artery Disease Risk

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit specific immunogenic double stranded RNA (dsRNA) molecules to prevent the dsRNA sensor, MDA5 ( IFIH1 ), activating an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). Here, we reveal in human atherosclerosis, smooth muscle cells (SMCs) express immunogenic dsRNA at markedly higher levels compared to all other cell types. We demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC). Loss of RNA editing through ADAR1 KD in HCASMC causes an ISG response characterized by upregulation of ISG genes (i.e. ISG15, IFI6, IFIH1 ) in addition to genes with known roles in SMC phenotypic transition and CAD risk (i.e. KLF4 , TCF21 , and ZEB2 ). ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro and in atherosclerosis prone mice. scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype. To further evaluate the role for SMC RNA editing in vivo , we generated a conditional SMC specific Adar1 KD mouse ( Adar1 flox/flox , Myh11 CreERT2 , ROSA tdT/+ , ApoE -/- ). At 2 weeks following SMC- Adar1 KD, we observe 50% mortality, where mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model ( Adar1 flox/flox , Cx40 CreERT2 ). scRNAseq of aortic tissue shows that SMC- Adar1 KD causes ISG activation throughout the vessel wall, SMCs show loss of mature SMC markers, and there is robust macrophage and T cell infiltration. Through evaluation of ligand-receptor interaction, we show that Ccl5:Ccr5 is the principal regulator of cell infiltration following SMC- Adar1 KD. Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.