Mouse Embryonic Cells Research Articles

Ziyin Bushen Fang improves Diabetic Osteoporosis by Inhibiting Autophagy and Oxidative Stress In vitro and In vivo.

Diabetic osteoporosis (DOP) belongs to the group of diabetes-induced secondary osteoporosis and is the main cause of bone fragility and fractures in many patients with diabetes. The aim of this study was to determine whether Ziyin Bushen Fang (ZYBSF) can improve DOP by inhibiting autophagy and oxidative stress. Type 1 diabetes mellitus (T1DM) was induced in rats using a high-fat high-sugar diet combined with streptozotocin. Micro-CT scanning was used to quantitatively observe changes in the bone microstructure in each group. Changes in the serum metabolites of DOP rats were analyzed using UHPLC-QTOF-MS. The DOP mouse embryonic osteoblast precursor cell model (MC3T3-E1) was induced using high glucose levels. After ZYBSF treatment, bone microstructure significantly improved. The bone mineral density, trabecular number, and trabecular thickness in the ZYBSF-M and ZYBSF-H groups significantly increased. After ZYBSF treatment, the femur structure of the rats was relatively intact, collagen fibers were significantly increased, and osteoporosis was significantly improved. A total of 1239 metabolites were upregulated and 1527 were downregulated in the serum of T1DM and ZYBSF-treated rats. A total of 20 metabolic pathways were identified. In cellular experiments, ZYBSF reduced ROS levels and inhibited the protein expression of LC3II / I, Beclin-1, and p-ERK. ZYBSF may improve DOP by inhibiting the ROS/ERK-induced autophagy signaling pathway.

Synthesis and comprehensive characterization with anticancer activity assessment of salicylaldehyde and 2-acetylphenol based chitosan thiosemicarbazones and their copper(II) complexes

Functionalization of chitosan as salicylaldehyde and 2-acetylphenol-based chitosan thiosemicarbazone involving amino group at C2 position of pyranose ring was confirmed by comprehensive characterization by spectroscopic and analytical ways. Both the low molecular weight (Mw) chitosan oligosaccharide (CS) (Mw < 3000 Da, degree of deacetylation (DDA) = 87 %) and the crab shell chitosan (CCS) (Mw = 350 kDa, DDA = 67 %) functionalized thiosemicarbazones behaved as ONS tridentate ligand to form the square planar copper(II) complexes with a chloride ion as an additional ligand to satisfy the co-ordination sphere. All the derivatives were found thermally stable till the commencement of chitosan backbone disruption at 200 °C. The MTT assay profiles showed a conspicuous enhancement in the in vitro antitumorigenic and anticancer activity of native chitosan upon its functionalization as phenolic carbaldehyde chitosan thiosemicarbazones. Further, the assays more often revealed a further lowering of cell viability with chitosan oligosaccharide derivatives in comparison to high molecular weight chitosan derivatives, and a lower viability of the MCF-7 cancer cells than the tumorigenic MDCK cells. Both the low Mw chitosan with high DDA and high Mw chitosan with low DDA, the chitosan-functionalized salicylaldehyde based thiosemicarbazones, and their copper(II) chitosan thiosemicarbazone complexes were found to have minimal toxicity with IC50 >600 μgmL−1 against the mouse embryonic normal fibroblast NIH3T3 cells. These results were indicatives of more protonation and enhanced anticancer activity of chitosan oligosaccharide derivatives, and preferential cytotoxicity of chitosan biomaterials against the tumorigenic and cancer cells with more negatively charged cell surfaces.

Molecular and Spatial Signatures of Mouse Embryonic Endothelial Cells at Single-Cell Resolution.

Mature endothelial cells (ECs) are heterogeneous, with subtypes defined by tissue origin and position within the vascular bed (ie, artery, capillary, vein, and lymphatic). How this heterogeneity is established during the development of the vascular system, especially arteriovenous specification of ECs, remains incompletely characterized. We used droplet-based single-cell RNA sequencing and multiplexed error-robust fluorescence in situ hybridization to define EC and EC progenitor subtypes from E9.5, E12.5, and E15.5 mouse embryos. We used trajectory inference to analyze the specification of arterial ECs (aECs) and venous ECs (vECs) from EC progenitors. Network analysis identified candidate transcriptional regulators of arteriovenous differentiation, which we tested by CRISPR (clustered regularly interspaced short palindromic repeats) loss of function in human-induced pluripotent stem cells undergoing directed differentiation to aECs or vECs (human-induced pluripotent stem cell-aECs or human-induced pluripotent stem cell-vECs). From the single-cell transcriptomes of 7682 E9.5 to E15.5 ECs, we identified 19 EC subtypes, including Etv2+Bnip3+ EC progenitors. Spatial transcriptomic analysis of 15 448 ECs provided orthogonal validation of these EC subtypes and established their spatial distribution. Most embryonic ECs were grouped by their vascular-bed types, while ECs from the brain, heart, liver, and lung were grouped by their tissue origins. Arterial (Eln, Dkk2, Vegfc, and Egfl8), venous (Fam174b and Clec14a), and capillary (Kcne3) marker genes were identified. Compared with aECs, embryonic vECs and capillary ECs shared fewer markers than their adult counterparts. Early capillary ECs with venous characteristics functioned as a branch point for differentiation of aEC and vEC lineages. Our results provide a spatiotemporal map of embryonic EC heterogeneity at single-cell resolution and demonstrate that the diversity of ECs in the embryo arises from both tissue origin and vascular-bed position. Developing aECs and vECs share common venous-featured capillary precursors and are regulated by distinct transcriptional regulatory networks.

RBL2 represses the transcriptional activity of Multicilin to inhibit multiciliogenesis

A core pathophysiologic feature underlying many respiratory diseases is multiciliated cell dysfunction, leading to inadequate mucociliary clearance. Due to the prevalence and highly variable etiology of mucociliary dysfunction in respiratory diseases, it is critical to understand the mechanisms controlling multiciliogenesis that may be targeted to restore functional mucociliary clearance. Multicilin, in a complex with E2F4, is necessary and sufficient to drive multiciliogenesis in airway epithelia, however this does not apply to all cell types, nor does it occur evenly across all cells in the same cell population. In this study we further investigated how co-factors regulate the ability of Multicilin to drive multiciliogenesis. Combining data in mouse embryonic fibroblasts and human bronchial epithelial cells, we identify RBL2 as a repressor of the transcriptional activity of Multicilin. Knockdown of RBL2 in submerged cultures or phosphorylation of RBL2 in response to apical air exposure, in the presence of Multicilin, allows multiciliogenesis to progress. These data demonstrate a dynamic interaction between RBL2 and Multicilin that regulates the capacity of cells to differentiate and multiciliate. Identification of this mechanism has important implications for facilitating MCC differentiation in diseases with impaired mucociliary clearance.

Selective induction of human renal interstitial progenitor-like cell lineages from iPSCs reveals development of mesangial and EPO-producing cells

Recent regenerative studies using human pluripotent stem cells (hPSCs) have developed multiple kidney-lineage cells and organoids. However, to further form functional segments of the kidney, interactions of epithelial and interstitial cells are required. Here we describe a selective differentiation of renal interstitial progenitor-like cells (IPLCs) from human induced pluripotent stem cells (hiPSCs) by modifying our previous induction method for nephron progenitor cells (NPCs) and analyzing mouse embryonic interstitial progenitor cell (IPC) development. Our IPLCs combined with hiPSC-derived NPCs and nephric duct cells form nephrogenic niche- and mesangium-like structures invitro. Furthermore, we successfully induce hiPSC-derived IPLCs to differentiate into mesangial and erythropoietin-producing cell lineages invitro by screening differentiation-inducing factors and confirm that p38 MAPK, hypoxia, and VEGF signaling pathways are involved in the differentiation of mesangial-lineage cells. These findings indicate that our IPC-lineage induction method contributes to kidney regeneration and developmental research.

Six3 and Six6 jointly control diverse target genes in multiple cell populations over developmental trajectories of mouse embryonic retinal progenitor cells.

How tissue-specific progenitor cells generate adult tissues is a puzzle in organogenesis. Using single-cell RNA sequencing of control and Six3 and Six6 compound-mutant mouse embryonic eyecups, we demonstrated that these two closely related transcription factors jointly control diverse target genes in multiple cell populations over the developmental trajectories of mouse embryonic retinal progenitor cells. In the Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) graph of control retinas, naïve retinal progenitor cells had two major trajectories leading to ciliary margin cells and retinal neurons, respectively. The ciliary margin trajectory was from naïve retinal progenitor cells in the G1 phase directly to ciliary margin cells, whereas the neuronal trajectory went through an intermediate neurogenic state marked by Atoh7 expression. Neurogenic retinal progenitor cells (Atoh7+) were still proliferative; early retinal neurons branched out from Atoh7+ retina progenitor cells in the G1 phase. Upon Six3 and Six6 dual deficiency, both naïve and neurogenic retinal progenitor cells were defective, ciliary margin differentiation was enhanced, and multi-lineage neuronal differentiation was disrupted. An ectopic neuronal trajectory lacking the Atoh7+ state led to ectopic neurons. Additionally, Wnt signaling was upregulated, whereas FGF signaling was downregulated. Notably, Six3 and Six6 proteins occupied the loci of diverse genes that were differentially expressed in distinct cell populations, and expression of these genes was significantly altered upon Six3 and Six6 dual deficiency. Our findings provide deeper insight into the molecular mechanisms underlying early retinal differentiation in mammals.

Analysis of Proliferation, Apoptosis, and Motility in Mouse Embryonic Melanocytic Precursor Cells.

In this chapter, we provide a method to purify and culture embryonic melanocytic stem cells that express green fluorescent protein in a cell-type specific manner. Isolation of melanocytic lineage cell populations that are >98% pure is accomplished through the use of GFP-based fluorescence activated cell sorting. We also provide a method to culture the purified melanoblasts and to analyze their proliferation, apoptosis, and motility properties.

Microcrystallization Effects in Borosilicate Bioactive Glasses: Controllable Release of Bioactive Elements and In Vitro Degradation Properties.

Borosilicate bioactive glasses exhibit excellent bioactivity and degradation properties; however, they suffer from the rapid release of bioactive elements at the initial stage of their degradation. Excessive local concentrations (such as those of B) can affect cell proliferation. Moreover, the degradation and mineralization ability of these glasses deteriorate at the later stages. Aiming to balance the release of bioactive elements during the whole process, herein, a borosilicate bioactive glass 18SiO2-6Na2O-8K2O-8MgO-22CaO-2P2O5-36B2O3 (mol%) was prepared using the melting method. Further, the effects of microcrystallization on the release of bioactive elements and in vitro degradation were studied. Results show that after heat treatment at temperatures over 620 °C, multiple microcrystalline phases, including Ca2SiO4, CaB2O4, and CaMgB2O5, form in the glass. The glass samples heat-treated within the range of 620-640 °C undergo appropriate devitrification degrees, decelerating the rate of pH increase of the immersion solution during the initial stage in comparison to those treated at lower temperatures. This results in a more continuous release of all bioactive elements and allows better control of the overall degradation. Contrarily, the more extensive devitrification degrees of glass samples heat-treated at higher temperatures reverse the pH increase and degradation trends. Since bone marrow mesenchymal stem cells and mouse embryonic osteoblast cells are pH-sensitive, inducing a suitable degree of devitrification proved to favor cell viability and enhance the mineralization capacity. Thus, different microcrystallization degrees provide new approaches for controlling the degradation and release of bioactive elements, resulting in the simultaneous enhancement of biosafety and bioactivity.

Transcription factor-mediated direct cellular reprogramming yields cell-type specific DNA methylation signature

Direct reprogramming, inducing the conversion of one type of somatic cell into another by the forced expression of defined transcription factors, is a technology with anticipated medical applications. However, due to the many unresolved aspects of the induction mechanisms, it is essential to thoroughly analyze the epigenomic state of the generated cells. Here, we performed comparative genome-wide DNA methylation analyses of mouse embryonic fibroblasts (MEFs) and cells composing organoids formed by intestinal stem cells (ISCs) or induced ISCs (iISCs) that were directly induced from MEFs. We found that the CpG methylation state was similar between cells forming ISC organoids and iISC organoids, while they differed widely from those in MEFs. Moreover, genomic regions that were differentially methylated between ISC organoid- and iISC organoid-forming cells did not significantly affect gene expression. These results demonstrate the accuracy and safety of iISC induction, leading to the medical applications of this technology.

Microtubule-dependent apical polarization of basement membrane matrix mRNAs in mouse epithelial cells

The basement membrane (BM) demarcating epithelial tissues undergoes rapid expansion to accommodate tissue growth and morphogenesis during embryonic development. To facilitate the secretion of bulky BM proteins, their mRNAs are polarized basally in the follicle epithelial cells of the Drosophila egg chamber to position their sites of production close to their deposition. In contrast, we observed the apical rather than basal polarization of all major BM mRNAs in the outer epithelial cells adjacent to the BM of mouse embryonic salivary glands using single-molecule RNA fluorescence in situ hybridization (smFISH). Moreover, electron microscopy and immunofluorescence revealed apical polarization of both the endoplasmic reticulum (ER) and Golgi apparatus, indicating that the site of BM component production was opposite to the site of deposition. At the apical side, BM mRNAs colocalized with ER, suggesting they may be co-translationally tethered. After microtubule inhibition, the BM mRNAs and ER became uniformly distributed rather than apically polarized, but they remained unchanged after inhibiting myosin II, ROCK, or F-actin, or after enzymatic disruption of the BM. Because Rab6 is generally required for Golgi-to-plasma membrane trafficking of BM components, we used lentivirus to express an mScarlet-tagged Rab6a in salivary gland epithelial cultures to visualize vesicle trafficking dynamics. We observed extensive bidirectional vesicle movements between Golgi at the apical side and the basal plasma membrane adjacent to the BM. Moreover, we showed that these vesicle movements depend on the microtubule motor kinesin-1 because very few vesicles remained motile after treatment with kinesore to compete for cargo-binding sites on kinesin-1. Overall, our work highlights the diverse strategies that different organisms use to secrete bulky matrix proteins: while Drosophila follicle epithelial cells strategically place their sites of BM protein production close to their deposition, mouse embryonic epithelial cells place their sites of production at the opposite end. Instead of spatial proximity, they use the microtubule cytoskeleton to mediate this organization as well as for the apical-to-basal transport of BM proteins.

Molecular Mechanism of the Asarum-Angelica Drug Pair in the Treatment of Periodontitis Based on Network Pharmacology and Experimental Verification.

(1) To examine the potential mechanism of the Asarum-Angelica drug pair against periodontitis and provide an experimental basis for the treatment of periodontitis with herbal medicine. (2) The core components and core targets of the Asarum-Angelica drug pair in the treatment of periodontitis were detected according to network pharmacology methods. Finally, the effect of the Asarum-Angelica drug pair on osteogenic differentiation was observed in mouse embryonic osteoblast precursor cells. (3) According to the results of network pharmacology, there are 10 potential active ingredients in the Asarum-Angelica drug pair, and 44 potential targets were obtained by mapping the targets with periodontitis treatment. Ten potential active ingredients, such as kaempferol and β-sitosterol, may play a role in treating periodontitis. Cell experiments showed that the Asarum-Angelica drug pair can effectively promote the expression of osteoblast markers alkaline phosphatase (ALP), Runt-related Transcription Factor 2 (RUNX2), and BCL2 mRNA and protein in an inflammatory environment (p < 0.05). (4) Network pharmacology effectively analyzed the molecular mechanism of Asarum-Angelica in the treatment of periodontitis, and the Asarum-Angelica drug pair can promote the differentiation of osteoblasts.

A remote atmospheric pressure plasma-assisted textile functionalization process on polymeric scaffolds for bone tissue engineering

When adopting polylactic acid (PLA) nonwoven for bone tissue engineering scaffolds, the inert and hydrophobic nature of PLA leads to poor biocompatibility and osteoconductivity, and limits the efficiency of applying bioceramic coatings, such as hydroxyapatite (HAp), for surface functionalization. To tackle these issues, a remote atmospheric pressure plasma (APP) system using a bespoke Pyrex chamber and acrylic acid monomer was designed and employed to functionalize the PLA via depositing carboxylic acid functional groups onto its surface. The remote APP showed being polymeric textile-suitable with an improved efficiency on not only immobilizing carboxylic acid functional groups on the PLA nonwoven than conventional systems but also the alternate soaking process for HAp deposition. Being compared with the neat PLA, the plasma-functionalized PLA exhibited a more hydrophilic surface (from 130.49 ± 1.28° to 117.40 ± 2.53°) with a 30-day stability (from 117.40 ± 2.53° to 119.40 ± 0.75°) and showed better biocompatibility (136 % to the neat PLA) with MC3T3-E1 mouse embryonic osteoblast cells after HAp deposition. The presence of carboxylic acid functional groups and HAp (Ca/P ratio of 1.7) was also identified using X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy and a scanning electron microscope with energy dispersive X-ray analysis.

Proteomic Screening Identifies Megakaryocyte Derived PF4/Cxcl4 As a Critical Driver of Myelofibrosis

Background: Despite advances in understanding the genomic landscape of Myeloproliferative Neoplasms (MPNs), the precise pathological mechanisms driving fibrotic progression in MPNs remain incompletely understood. Previous research has shown that Megakaryocytes (Mks) play a role in MPN pathology by promoting inflammation and extracellular matrix (ECM) deposition by activated stromal cells. However, the specific factors derived from Mks that contribute to the inflammatory milieu and myelofibrosis progression are not well defined. Aims: To investigate the events that drive bone marrow (BM) fibrosis due to abnormal megakaryocyte-stromal crosstalk, we employed mass spectrometry-based proteomics in a myelofibrosis-like mouse model. Our goal was to identify differentially expressed proteins involving Mks, platelets and BM cells in fibrotic versus non-fibrotic conditions. Methods: We induced myelofibrosis in mice through repeated injections of supra-pharmacological doses of the thrombopoietin-mimetic Romiplostim (TPO high). Subsequently, we isolated Mks, BM cells and platelets from TPO high and control mice (injected with saline) and analyzed protein extracts using LC-MS/MS assay. Cytokine levels in plasma and BM cell-free fluids of treated mice were assessed using cytokine arrays. To evaluate fibrosis-related markers, we analyzed the expression of myofibroblasts markers (a-SMA, Vimentin) and ECM synthesis (Fibronectin, Type III Collagen) in mouse embryonic fibroblasts (mEFs) and human BM mesenchymal stromal cells after stimulation with recombinant PF4 using western blot. mEFs were co-cultured with BM-derived Mks in direct and indirect co-culture systems in the presence of increasing TPO concentrations. In some experiments, Mks were transfected with siRNA specifically targeting CXCL4 gene or control siRNA before co-culture with mEFs. Additionally, we quantified plasma PF4/Cxcl4 levels using ELISA assay in a large cohort of MPN patients and normal controls. We also sorted CD41/CD61 Mks from BM aspirates of MPN patients and evaluated CXCL4 mRNA levels through qPCR. Results: We observed that signaling pathways related to cytoskeletal reorganization, cell adhesion and inflammation, were commonly activated in Mks, platelets and BM cells of TPO high mice compared to control mice. The MSP-RON signaling pathway and the Unfolded Protein Response were specifically activated in Mks and platelets, respectively. Among the differentially expressed proteins,the chemokine PF4/Cxcl4 was up regulated exclusively in the proteasomes of the TPO high mice. We confirmed Increased levels of PF4/Cxcl4 in plasma and BM cell-free fluids of fibrotic mice using cytokine arrays. In vitro stimulation of BM-derived Mks with high TPO concentrations resulted in increased synthesis and secretion of PF4/Cxcl4. Recombinant PF4/Cxcl4 was rapidly internalized through surface glycosaminoglycans by mEFs and human BM mesenchymal stromal cells, leading to myofibroblast differentiation. Importantly, we demonstrated that these mechanisms were interconnected during Mk-mEF cross-talk in co-culture systems with high TPO concentrations. Genetic down-regulation of CXCL4 in Mks, prior to co-culture in TPO-saturated conditions, mitigated the profibrotic phenotype of mEFs in terms of ECM synthesis and myofibroblast markers expression. Finally, we found higher plasma levels of circulating PF4/Cxcl4 and increased expression in BM-sorted Mks in patients with MPNs. Conclusions: Our findings identify a crucial role of Mk derived PF4 in the fibrosis progression of MPNs, providing further support for the potential therapeutic strategy of neutralizing PF4.

Design, synthesis and evaluation of novel pyrido fused piperazine derivatives: In vitro cytotoxicity study over various cancer cell lines

In the present work, a series of novel pyrido bearing a fused piperazine ring derivatives has been synthesized via suzuki and acid–amine coupling reactions. The synthesized compounds were screened for their in vitro cytotoxic activity against nine different cancer cell lines such as U-251, Panc-1, MCF-7, HepG2, DU-145, A549, 786-O, OVCAR3 and HCT-116. Synthesized compound also screened under normal cell lines like human lungs normal cell line (WI38) and Mouse embryonic normal cell line (NIH3T3) to evaluate safety of synthesized compounds. The bioassay results indicates that synthesized compounds (cpd-S10, cpd-12, cpd-19, cpd-32, cpd-33, cpd-36 and cpd-38) to be moderately effective towards various cancer cell lines (DU-145, PanC1, MCF7, U-251, HCT-116, 786-O, HepG2, A549, OVCAR-3) in the in vitro testing.

Cooperative Response to Endocardial Notch Reveals Interaction With Hippo Pathway.

The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors. We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential. We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the endocardium during cardiac valve development. Sequential Notch-dependent transcriptional regulation in the embryonic endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.

HMGA2 directly mediates chromatin condensation in association with neuronal fate regulation

Identification of factors that regulate chromatin condensation is important for understanding of gene regulation. High-mobility group AT-hook (HMGA) proteins 1 and 2 are abundant nonhistone chromatin proteins that play a role in many biological processes including tissue stem-progenitor cell regulation, but the nature of their protein function remains unclear. Here we show that HMGA2 mediates direct condensation of polynucleosomes and forms droplets with nucleosomes. Consistently, most endogenous HMGA2 localized to transposase 5– and DNase I–inaccessible chromatin regions, and its binding was mostly associated with gene repression, in mouse embryonic neocortical cells. The AT-hook 1 domain was necessary for chromatin condensation by HMGA2 in vitro and in cellulo, and an HMGA2 mutant lacking this domain was defective in the ability to maintain neuronal progenitors in vivo. Intrinsically disordered regions of other proteins could substitute for the AT-hook 1 domain in promoting this biological function of HMGA2. Taken together, HMGA2 may regulate neural cell fate by its chromatin condensation activity.

Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury

The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and β-Catenin signaling cascade, but only Erk inhibitors reversed RSPO3-induced neuroprotection against OGD/R. In mouse embryonic fibroblasts (MEFs) and neuronal cells, RSPO3-induced LGR4-Gab1-Gαi1/3 association was required for Erk activation, and either silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. In mice, middle cerebral artery occlusion (MCAO) increased RSPO3 expression and Erk activation in ischemic penumbra brain tissues. Endothelial knockdown or knockout of RSPO3 inhibited Erk activation in the ischemic penumbra brain tissues and increased MCAO-induced cerebral ischemic injury in mice. Conversely, endothelial overexpression of RSPO3 ameliorated MCAO-induced cerebral ischemic injury. We conclude that RSPO3 activates Gαi1/3-Erk signaling to protect neuronal cells from ischemia/reperfusion injury.

OR18-01 Massively Parallel Functional Assay Of Extracellular Insulin Receptor Variants

Abstract Disclosure: V. Aslanzadeh: None. G. Brierley: None. G. Kudla: None. R.K. Semple: None. Loss-of-function mutations in the gene for the human insulin receptor, INSR, cause either autosomal dominant or autosomal recessive severe insulin resistance. Biallelic mutations produce extreme insulin resistance which is commonly fatal in infancy or childhood. A wide and growing number of mutations have been associated with disease, with few recurring mutations, and only a small subset have been studied functionally to date. Discriminating loss-of-function pathogenic alleles from insignificant variants is an important barrier to diagnosis and translational studies of novel candidate treatments. Such candidate therapies include a variety of antibody, peptide and small molecule ligands that activate the receptor in a manner distinct to insulin. These show potential to activate mutant receptors that are expressed but show impaired insulin binding or responsiveness. To address this barrier to translation we have now used massively multiplexed, Flow-Seq-based assays simultaneously to determine the effects of 14,000 human INSR extracellular domain missense variants. We assayed receptor cell surface expression, insulin binding and signal transduction using heterologous expression of mutants in mouse embryonic cells in which endogenous Insr and Igf1r genes had been knocked out or knocked down. Preliminary analysis reliably discriminates A. variants that reduce or abolish receptor cell surface expression when measured with two different monoclonal anti-receptor antibodies B. variants with preserved cell surface expression but selectively impaired binding of insulin and C. an overlapping group of variants with severely impaired signalling response to insulin but preserved response to antibody. Our assay confirmed previously studied attributes of a panel of disease-causing variants, and shows strong agreement with prior alanine scanning mutagenesis studies and structural predictions. We thus provide a rich sequence-function map for INSR variant interpretation. A prediction tool trained on known pathogenic INSR alleles is currently being developed for access by the community both to accelerate confident molecular diagnosis of newly identified rare INSR variants, and also to aid stratification of mutations according to potential for activation by non canonical INSR ligands. Presentation: Saturday, June 17, 2023

Inhibition of Cxcr4 Disrupts Mouse Embryonic Palatal Mesenchymal Cell Migration and Induces Cleft Palate Occurrence.

Many processes take place during embryogenesis, and the development of the palate mainly involves proliferation, migration, osteogenesis, and epithelial-mesenchymal transition. Abnormalities in any of these processes can be the cause of cleft palate (CP). There have been few reports on whether C-X-C motif chemokine receptor 4 (CXCR4), which is involved in embryonic development, participates in these processes. In our study, the knockdown of Cxcr4 inhibited the migration of mouse embryonic palatal mesenchymal (MEPM) cells similarly to the use of its inhibitor plerixafor, and the inhibition of cell migration in the Cxcr4 knockdown group was partially reversed by supplementation with C-X-C motif chemokine ligand 12 (CXCL12). In combination with low-dose retinoic acid (RA), plerixafor increased the incidence of cleft palates in mice by decreasing the expression of Cxcr4 and its downstream migration-regulating gene Rac family small GTPase 1 (RAC1) mediating actin cytoskeleton to affect lamellipodia formation and focal complex assembly and ras homolog family member A (RHOA) regulating the actin cytoskeleton to affect stress fiber formation and focal complex maturation into focal adhesions. Our results indicate that the disruption of cell migration and impaired normal palatal development by inhibition of Cxcr4 expression might be mediated through Rac1 with RhoA. The combination of retinoic acid and plerixafor might increase the incidence of cleft palate, which also provided a rationale to guide the use of the drug during conception.

Abstract P2096: Characterization Of Unique Dystrophin Isoforms In Epicardial Cells

Duchenne muscular dystrophy (DMD) is caused by mutations that abolish dystrophin expression. Dilated cardiomyopathy, the leading cause of death in DMD patients, is characterized by the development of severe subepicardial fibrosis that precedes cardiac dysfunction. While the cellular mechanisms governing this process remain unknown, a recent study in mdx/mTR mice suggested that inflammation, in response to cardiomyocyte damage caused by dystrophin deficiency, activates the epicardium to undergo epithelial-to-mesenchymal transition into fibroblasts, thereby contributing to subepicardial fibrosis. Whether dystrophin plays an epicardial-autonomous role in this process has not been investigated. The DMD gene produces at least eight dystrophin isoforms from seven independent, tissue-specific promoters ( Fig. 1 ). We used an immortalized mouse embryonic epicardial cell line to determine which isoforms are present. We found transcripts for the full-length muscle isoform of dystrophin (Dp427m) in addition to the ubiquitous, more abundant Dp71 and Dp40 isoforms. RT-PCR and western blot analysis revealed the presence of three different Dp71 splice variants. Immunofluorescence and subcellular fractionation determined that Dp71 localizes primarily to the nucleus. Studies are ongoing to understand the role of Dp71 in epicardial cell proliferation and gene expression, as well as defining changes in dystrophin expression during the transition from the activated epicardium to myofibroblasts. Further investigation is needed to determine the role of these shorter isoforms in DMD pathology and how gene editing of full-length dystrophin may impact their function.