Cell Phenotype Research Articles

Biomimetic “nano-spears” for CAFs-targeting: splintered three “shields” with enhanced cisplatin anti-TNBC efficiency

The treatment dilemma of triple-negative breast cancer (TNBC) revolves around drug resistance and metastasis. Cancer-associated fibroblasts (CAFs) contribute to cisplatin (Cis) resistance and further metastasis in TNBC, making TNBC a difficult-to-treat disease. The dense stromal barrier which restricts drug delivery, invasive phenotype of tumor cells, and immunosuppressive tumor microenvironment (TME) induced by CAFs serve as three “shields” for TNBC against Cis therapy. Here, we designed a silybin-loaded biomimetic nanoparticle coated with anisamide-modified red blood cell membrane (ARm@SNP) as a “nanospear” for CAFs-targeting, which could shatter the “shields” and significantly exhibit inhibitory effect on 4T1 cells in combination with Cis both in vitro and in vivo. The ARm@SNP/Cis elicited 4T1 tumor growth arrest and destroyed three “shields” as follows: disintegrating the stromal barrier by inhibiting blood vessels growth and the expression of fibronectin; decreasing 4T1 cell invasion and metastasis by affecting the TGF-β/Twist/EMT pathway which impeded EMT activation; reversing the immunosuppressive microenvironment by increasing the activity and infiltration of immunocompetent cells. Based on CAFs-targeting, ARm@SNP reversed the resistance of Cis, remodeled the TME and inhibited invasion and metastasis while significantly improving the therapeutic effect of Cis on 4T1 tumor-bearing mice, providing a promising approach for treating intractable TNBC.

The Histone Demethylase JMJD3 Regulates Smooth Muscle Cell Phenotype during Macrophage-mediated Inflammation

The Histone Demethylase JMJD3 Regulates Smooth Muscle Cell Phenotype during Macrophage-mediated Inflammation

Coarsened Mixtures of Hierarchical Skew Normal Kernels for Flow and Mass Cytometry Analyses

Cytometry is the standard multi-parameter assay for measuring single cell phenotype and functionality. It is commonly used for quantifying the relative frequencies of cell subsets in blood and disaggregated tissues. A typical analysis of cytometry data involves cell classification—that is, the identification of cell subgroups in the sample—and comparisons of the cell subgroups across samples or conditions. While modern experiments often necessitate the collection and processing of samples in multiple batches, analysis of cytometry data across batches is challenging because differences across samples may occur due to either true biological variation or technical reasons such as antibody lot effects or instrument optics across batches. Thus a critical step in comparative analyses of multi-sample cytometry data—yet missing in existing automated methods for analyzing such data—is cross-sample calibration, whose goal is to align corresponding cell subsets across multiple samples in the presence of technical variations, so that biological variations can be meaningfully compared. We introduce a Bayesian nonparametric hierarchical modeling approach for accomplishing both calibration and cell classification simultaneously in a unified probabilistic manner. Three important features of our method make it particularly effective for analyzing multi-sample cytometry data: a nonparametric mixture avoids prespecifying the number of cell clusters; a hierarchical skew normal kernel that allows flexibility in the shapes of the cell subsets and cross-sample variation in their locations; and finally the “coarsening” strategy makes inference robust to departures from the model not captured by the skew normal kernels. We demonstrate the merits of our approach in simulated examples and carry out a case study in the analysis of a multi-sample cytometry data set. We provide an R package for our method.

Reduction of Abdominal Aortic Aneurysm Rupture by Modulating Smooth Muscle Cell Phenotype Switching

Reduction of Abdominal Aortic Aneurysm Rupture by Modulating Smooth Muscle Cell Phenotype Switching

KIF23 promotes cervical cancer progression via inhibiting NLRP3-mediated pyroptosis.

Cervical cancer (CC), closely linked to persistent human papillomavirus infection, represents a major health problem for women worldwide. The objective of this study is to elucidate KIF23's role in the development of CC and its regulatory mechanism. The bioinformatics methods were utilized to extract pyroptosis-associated differentially expressed genes (DEGs) and pivot genes from the GSE9750 and GSE63678 datasets, followed by immune infiltration analysis and quantification of these genes' expression. The effects of kinesin family member 23 (KIF23) were verified through functional experiments invitro and a mouse xenograft model. The NLPR3 activator, nigericin, was applied for further analyzing the potential regulatory mechanism of KIF23 in CC. A total of 8 pyroptosis-related DEGs were screened out, among which 4 candidate core genes were identified as candidate hub genes and confirmed upregulation in CC tissues and cells. These genes respectively showed a positive correlation with the infiltration of distinct immune cells or tumor purity. Downregulation of KIF23 could suppress the proliferation, migration, and invasion abilities in CC cells and tumorigenesis through enhancing pyroptosis. Conversely, KIF23 overexpression accelerated the malignant phenotypes of CC cells and inhibited pyroptosis activation, which was blocked by nigericin treatment. KIF23 may play an oncogenic role in CC progression via inhibition of the NLRP3-mediated pyroptosis pathway.

Dissecting causal relationships between immune cells, plasma metabolites, and COPD: a mediating Mendelian randomization study

ObjectiveThis study employed Mendelian Randomization (MR) to investigate the causal relationships among immune cells, COPD, and potential metabolic mediators.MethodsUtilizing summary data from genome-wide association studies, we analyzed 731 immune cell phenotypes, 1,400 plasma metabolites, and COPD. Bidirectional MR analysis was conducted to explore the causal links between immune cells and COPD, complemented by two-step mediation analysis and multivariable MR to identify potential mediating metabolites.ResultsCausal relationships were identified between 41 immune cell phenotypes and COPD, with 6 exhibiting reverse causality. Additionally, 21 metabolites were causally related to COPD. Through two-step MR and multivariable MR analyses, 8 cell phenotypes were found to have causal relationships with COPD mediated by 8 plasma metabolites (including one unidentified), with 1-methylnicotinamide levels showing the highest mediation proportion at 26.4%.ConclusionWe have identified causal relationships between 8 immune cell phenotypes and COPD, mediated by 8 metabolites. These findings contribute to the screening of individuals at high risk for COPD and offer insights into early prevention and the precocious diagnosis of Pre-COPD.

Multiple sclerosis patient-derived spontaneous B cells have distinct EBV and host gene expression profiles in active disease.

Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4+ T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.

Harnessing Metformin’s Immunomodulatory Effects on Immune Cells to Combat Breast Cancer

Metformin, a medication known for its anti-glycemic properties, also demonstrates potent immune system activation. In our study, using a 4T1 breast cancer model in BALB/C WT mice, we examined metformin’s impact on the functional phenotype of multiple immune cells, with a specific emphasis on natural killer T (NKT) cells due to their understudied role in this context. Metformin administration delayed the appearance and growth of carcinoma. Furthermore, metformin increased the percentage of IFN-γ+ NKT cells, and enhanced CD107a expression, as measured by MFI, while decreasing PD-1+, FoxP3+, and IL-10+ NKT cells in spleens of metformin-treated mice. In primary tumors, metformin increased the percentage of NKp46+ NKT cells and increased FasL expression, while lowering the percentages of FoxP3+, PD-1+, and IL-10-producing NKT cells and KLRG1 expression. Activation markers increased, and immunosuppressive markers declined in T cells from both the spleen and tumors. Furthermore, metformin decreased IL-10+ and FoxP3+ Tregs, along with Gr-1+ myeloid-derived suppressor cells (MDSCs) in spleens, and in tumor tissue, it decreased IL-10+ and FoxP3+ Tregs, Gr-1+, NF-κB+, and iNOS+ MDSCs, and iNOS+ dendritic cells (DCs), while increasing the DCs quantity. Additionally, increased expression levels of MIP1a, STAT4, and NFAT in splenocytes were found. These comprehensive findings illustrate metformin’s broad immunomodulatory impact across a variety of immune cells, including stimulating NKT cells and T cells, while inhibiting Tregs and MDSCs. This dynamic modulation may potentiate its use in cancer immunotherapy, highlighting its potential to modulate the tumor microenvironment across a spectrum of immune cell types.

Three Dimensional Porous Binary Composites of Collagen, Elastin and Fibrin Proteins Orchestrate Adipose Tissue Regeneration.

The development of an optimal healthcare biomaterial for targeted tissue regeneration poses a significant challenge. Our current objective is to advance the development of a specialised biomaterial that can effectively facilitate the regeneration of adipose tissue. In prior studies, the assessment of collagen, elastin, and fibrin unary scaffolds has been conducted. However, it is important to note that native adipose tissue is comprised of a diverse array of extracellular matrix (ECM) constituents. To mimic this behaviour we fabricated binary compositions of collagen, elastin, and fibrin in a 1:1 ratio, resulting in the formation of Col/Ela, Col/Fib, and Ela/Fib composites through a customised fabrication procedure. The physical properties of these scaffolds were comprehensively analysed using a range of material characterisation techniques. Additionally, the biological properties of the scaffolds were investigated by examining the survival, proliferation, and phenotype of adipose-derived stem cells. Subsequently, the aforementioned binary scaffolds were introduced into a live rodent model for a duration of 28 days. Following this period, the explants were subjected to analysis through X-ray microtomography, histology, and immunohistochemistry in order to evaluate their immunocompatibility, integration, and adipogenesis. The findings of the study demonstrated that the utilisation of binary combinations of Col/Ela, Col/Fib, and Ela/Fib had a discernible impact on the physical and biological characteristics of the scaffolds. Nevertheless, Ela/Fib exhibited characteristics that made it a suitable candidate for adipogenesis due to its notable upregulation of caveolin-1 expression in both acellular and cellular cohorts. The combination of two natural polymers in this cell-material interaction has significantly enhanced our comprehension of adipogenesis. This article is protected by copyright. All rights reserved.

Construction and Identification of a Human Colorectal Adenoma Epithelial Cell Line by SV40 Large T-Antigen Transfection.

Colorectal adenoma represents the critical step in the development of colorectal cancer. The establishment of an immortalized epithelial cell line of colorectal adenoma of human origin would provide a tool for studying the mechanism of precancerous lesions, screening the efficacy of novel drugs, and constructing in vivo disease models. Currently, there is no commercially available stable supply of epithelial cells from precancerous lesions. This study aimed to establish a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection. Simian vacuolating virus 40(SV40), SV40-LT overexpressed lentivirus vector, was transfected into primary human colorectal adenomatous polyp epithelial cells. The transfected cells were screened, and the screened cells were amplified to obtain the epithelial cell line: IHCRA- CELL. The cells were identified by morphological observation, cell proliferation, Quantitative real-time PCR (qPCR), and Short Tandem Repeats (STR) experiments. Morphologically, the cells showed epithelial-like characteristics, such as polygon shape, desmosomes mitochondria, and strong positive keratin staining. There was no significant difference between the transfected cells and the primary cells. Through the STR identification experiment, no matching cell lines were found in the cell lines retrieval. We successfully established a natural LHPP low-expressing precancerous epithelial cell line by SV40-LT antigen gene transfection, which has been patented and is now preserved in the Chinese Typical Culture Preservation Center. It was verified that the transformed cells maintained the phenotype and biological characteristics of epithelial cells. This cell line can be used to study the mechanism of precancerous lesions, screen the efficacy of novel drugs, and construct in vivo disease models.

Tubular insulin-induced gene 1 deficiency promotes NAD+ consumption and exacerbates kidney fibrosis.

Profibrotic proximal tubules (PT) were identified as a unique phenotype of proximal tubule cells (PTCs) in renal fibrosis by single-cell RNA sequencing (scRNA-seq). Controlling the process of renal fibrosis requires understanding how to manage the S1 subset's branch to the S3 subset rather than to the profibrotic PT subset. Insulin-induced gene 1 (Insig1) is one of the branch-dependent genes involved in controlling this process, although its role in renal fibrosis is unknown. Here, we discovered that tubular Insig1 deficiency, rather than fibroblast Insig1 deficiency, plays a detrimental role in the pathogenesis of renal fibrosis in vivo and in vitro. Overexpression of Insig1 profoundly inhibited renal fibrosis. Mechanistically, Insig1 deletion in PTCs boosted SREBP1 nuclear localization, increasing Aldh1a1 transcriptional activity, causing excessive NAD+ consumption and ER enlargement, as well as accelerating renal fibrosis. We also identified nicardipine as a selective inhibitor of Aldh1a1, which could restore NAD+ and maintain ER homeostasis, as well as improve renal fibrosis. Together, our findings support tubular Insig1 as a new therapeutic target for chronic kidney disease (CKD).

Metadherin-driven promotion of cancer stem cell phenotypes and its effect on immunity in hepatocellular carcinoma

Metadherin-driven promotion of cancer stem cell phenotypes and its effect on immunity in hepatocellular carcinoma

SETBP1 activation upon MDM4-enhanced ubiquitination of NR3C1 triggers dissemination of colorectal cancer cells.

Colorectal cancer (CRC) presents a growing concern globally, marked by its escalating incidence and mortality rates, thus imposing a substantial health burden. This investigation delves into the role of nuclear receptor subfamily 3 group C member 1 (NR3C1) in CRC metastasis and explores the associated mechanism. Through a comprehensive bioinformatics analysis, NR3C1 emerged as a gene with diminished expression levels in CRC. This finding was corroborated by observations of a low-expression pattern of NR3C1 in both CRC tissues and cells. Furthermore, experiments involving NR3C1 knockdown revealed an exacerbation of proliferation, migration, and invasion of CRC cells in vitro. Subsequent assessments in mouse xenograft tumor models, established by injecting human HCT116 cells either through the tail vein or at the cecum termini, demonstrated a reduction in tumor metastasis to the lung and liver, respectively, upon NR3C1 knockdown. Functionally, NR3C1 (glucocorticoid receptor) suppressed SET binding protein 1 (SETBP1) transcription by binding to its promoter region. Notably, mouse double minute 4 (MDM4) was identified as an upstream regulator of NR3C1, orchestrating its downregulation via ubiquitination-dependent proteasomal degradation. Further investigations unveiled that SETBP1 knockdown suppressed migration and invasion, and epithelial to mesenchymal transition of CRC cells, consequently impeding in vivo metastasis in murine models. Conversely, upregulation of MDM4 exacerbated the metastatic phenotype of CRC cells, a propensity mitigated upon additional upregulation of NR3C1. In summary, this study elucidates a cascade wherein MDM4-mediated ubiquitination of NR3C1 enables the transcriptional activation of SETBP1, thereby propelling the dissemination of CRC cells.

Interleukin-1 regulates follicular T cells during the germinal center reaction

IntroductionAntibody production and the generation of memory B cells are regulated by T follicular helper (Tfh) and T follicular regulatory (Tfr) cells in germinal centers. However, the precise role of Tfr cells in controlling antibody production is still unclear. We have previously shown that both Tfh and Tfr cells express the IL-1R1 agonist receptor, whereas only Tfr cells express the IL-1R2 decoy and IL-1Ra antagonist receptors. We aimed to investigate the role of IL-1 receptors in the regulation of B cell responses by Tfh and Tfr.MethodsWe generated mice with IL-1 receptors inactivated in Tfh or Tfr and measured antibody production and cell activation after immunisation.ResultsWhile IL-1β levels are increased in the draining lymph node after immunisation, antigen-specific antibody levels and cell phenotypes indicated that IL-1β can activate both Tfh and Tfr cells through IL-1R1 stimulation. Surprisingly, expression of IL-1R2 and IL-1Ra on Tfr cells does not block IL-1 activation of Tfh cells, but rather prevents IL-1/IL-1R1-mediated early activation of Tfr cells. IL-1Rs also regulate the antibody response to autoantigens and its associated pathophysiology in an experimental lupus model.DiscussionCollectively, our results show that IL-1 inhibitory receptors expressed by Tfr cells prevent their own activation and suppressive function, thus licensing IL-1-mediated activation of Tfh cells after immunisation. Further mechanistic studies should unravel these complex interactions between IL-1β and follicular helper and regulatory T cells and provide new avenues for therapeutic intervention.

Deposited footprints let cells switch between confined, oscillatory, and exploratory migration

For eukaryotic cells to heal wounds, respond to immune signals, or metastasize, they must migrate, often by adhering to extracellular matrix (ECM). Cells may also deposit ECM components, leaving behind a footprint that influences their crawling. Recent experiments showed that some epithelial cell lines on micropatterned adhesive stripes move persistently in regions they have previously crawled over, where footprints have been formed, but barely advance into unexplored regions, creating an oscillatory migration of increasing amplitude. Here, we explore through mathematical modeling how footprint deposition and cell responses to footprint combine to allow cells to develop oscillation and other complex migratory motions. We simulate cell crawling with a phase field model coupled to a biochemical model of cell polarity, assuming local contact with the deposited footprint activates Rac1, a protein that establishes the cell's front. Depending on footprint deposition rate and response to the footprint, cells on micropatterned lines can display many types of motility, including confined, oscillatory, and persistent motion. On two-dimensional (2D) substrates, we predict a transition between cells undergoing circular motion and cells developing an exploratory phenotype. Small quantitative changes in a cell's interaction with its footprint can completely alter exploration, allowing cells to tightly regulate their motion, leading to different motility phenotypes (confined vs. exploratory) in different cells when deposition or sensing is variable from cell to cell. Consistent with our computational predictions, we find in earlier experimental data evidence of cells undergoing both circular and exploratory motion.

Integrating phylogenies into single-cell RNA sequencing analysis allows comparisons across species, genes, and cells.

Comparisons of single-cell RNA sequencing (scRNA-seq) data across species can reveal links between cellular gene expression and the evolution of cell functions, features, and phenotypes. These comparisons evoke evolutionary histories, as depicted by phylogenetic trees, that define relationships between species, genes, and cells. This Essay considers each of these in turn, laying out challenges and solutions derived from a phylogenetic comparative approach and relating these solutions to previously proposed methods for the pairwise alignment of cellular dimensional maps. This Essay contends that species trees, gene trees, cell phylogenies, and cell lineages can all be reconciled as descriptions of the same concept-the tree of cellular life. By integrating phylogenetic approaches into scRNA-seq analyses, challenges for building informed comparisons across species can be overcome, and hypotheses about gene and cell evolution can be robustly tested.

The Loss of an Orphan Nuclear Receptor NR2E3 Augments Wnt/β-catenin Signaling via Epigenetic Dysregulation that Enhances Sp1-β catenin-p300 Interactions in Hepatocellular Carcinoma.

The orphan nuclear receptor NR2E3 (Nuclear receptor subfamily 2 group E, Member 3) is an epigenetic player that modulates chromatin accessibility to activate p53 during liver injury. Nonetheless, a precise tumor suppressive and epigenetic role of NR2E3 in hepatocellular carcinoma (HCC) development remains unclear. HCC patients expressing low NR2E3 exhibit unfavorable clinical outcomes, aligning with heightened activation of the Wnt/β-catenin signaling pathway. The murine HCC models utilizing NR2E3 knockout mice consistently exhibits accelerated liver tumor formation accompanied by enhanced activation of Wnt/β-catenin signaling pathway and inactivation of p53 signaling. At cellular level, the loss of NR2E3 increases the acquisition of aggressive cancer cell phenotype and tumorigenicity and upregulates key genes in the WNT/β-catenin pathway with increased chromatin accessibility. This event is mediated through increased formation of active transcription complex involving Sp1, β-catenin, and p300, a histone acetyltransferase, on the promoters of target genes. These findings demonstrate that the loss of NR2E3 activates Wnt/β-catenin signaling at cellular and organism levels and this dysregulation is associated with aggressive HCC development and poor clinical outcomes. In summary, NR2E3 is a novel tumor suppressor with a significant prognostic value, maintaining epigenetic homeostasis to suppress the Wnt/β-catenin signaling pathway that promotes HCC development.

#2052 Reciprocal interplay between miR-155-5p and SOCS1 in the development of renal and vascular damage in experimental diabetes

Abstract Background and Aims Chronic kidney disease and atherosclerosis are common vascular complication of diabetes and a global health issue. Hyperglycemia, dyslipidemia, and cytokines activate Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway to induce mediators of inflammation and oxidative stress, which are critical events in the progression of renal and vascular damage in diabetic patients. MicroRNAs are small, non-coding molecules and key regulators of gene expression. There is evidence of microRNA-155 (miR-155) involvement in diabetes complications, but the underlying mechanisms are unclear. In this study, gain- and loss-of-function experiments were applied to investigate the interplay between miR-155-5p and suppressor of cytokine signaling 1 (SOCS1) in the regulation of JAK/STAT pathway during renal and vascular injury in diabetes. Method Mesangial, tubuloepithelial, and vascular smooth muscle cells were transfected with miR-155-5p mimic or inhibitor, SOCS1 expression plasmid or siRNA, before stimulation with cytokines or high-glucose. Apolipoprotein E deficient mice with streptozotocin-induced diabetes were treated with either SOCS1-encoding adenovirus or miR-155-5p inhibitor. Samples were analyzed for the expression of miR-155-5p, SOCS1, and mediators of renal and vascular damage. Results In diabetic mice, the expression of miR-155-5p correlated inversely with SOCS1 levels and positively with markers of renal dysfunction, atherosclerosis, inflammation and oxidative stress. In renal cells, transfection with miR-155-5p mimic downregulated SOCS1, activated STAT1 and cytokine expression, and stimulated cell proliferation and migration. Mimic transfection also promoted the transition from contractile to synthetic phenotype in vascular smooth muscle cells. Conversely, both miR-155-5p antagonism and SOCS1 overexpression protected cells from inflammatory damage. In vivo SOCS1 gene delivery in diabetic mice decreased miR-155-5p and prevented kidney and vascular injury. Finally, therapeutic inhibition of miR-155-5p alleviates albuminuria, renal inflammation and fibrosis in diabetic mice, and also reduced atherosclerosis burden and improved plaque stability. Conclusion The modulation of miR-155/SOCS1 axis protects against diabetic renal and vascular damage in mice, thus highlighting its potential as therapeutic target for chronic complications of diabetes.

#2695 A Polycystin-1-Bicaudal C1 complex regulates focal adhesion function through integrin beta-1, F-actin organisation and local translation in HKE cells

Abstract Background and Aims Variants in Bicc1, result in a recessive murine PKD phenotype (bpk, jcpk) by uncertain mechanisms. In a recent study, we reported genetic and biochemical evidence of novel interactions between PKD1, PKD2 and BICC1 in Xenopus, mouse and man suggesting that BICC1 is a modifier of the ADPKD phenotype (submitted). In this study, our major aim was to investigate the molecular mechanisms underlying cystogenesis that result from loss of the normal interaction between the PKD1 protein, Polycystin-1 (PC1), a cell surface receptor and Bicaudal C1 (BICC1), an RNA-binding protein. Methods Isogenic PKD1 and BICC1 CRISP-Cas9 KO cells were generated from a conditionally-immortalised wild-type human proximal tubular line (UCL93); RNA-seq was performed in PKD1 and BICC1 KO cells to identify commonly altered genes and pathways; MS pull-down was conducted in HEK293 cells with BICC1-HA to identify BICC1 interacting proteins; cellular phenotypes in normal and KO cells were assessed through adhesion and migration assays, alongside Traction Force Microscopy (TFM); proximal ligation assays (PLA) to confirm protein-protein interactions; single-molecule FISH (smFISH) to localize mRNA transcripts; puromycin labeling with a proximity ligation assay (Puro-PLA) to detect sites of translation of specific mRNAs. Results Consistent with a functional interaction, loss of PC1 resulted in lower BICC1 levels and vice-versa in single KO (SKO) cells. In migrating cells, BICC1 was expressed at the leading edge where it co-localised with β1-integrin (ITGB1) by PLA, suggesting enrichment at focal adhesion sites (FA). BICC1 KO cells had no cilia phenotype but instead displayed striking disorganization of stress fibres and aster-like structures, features which could be completely rescued by lentiviral Bicc1-HA re-expression. Gene set enrichment and pathway analysis confirmed that F-actin organization and actin-associated proteins (AAP) were commonly dysregulated in SKO cells. BICC1 was also found to interact ITGB1, several components of an integrin-associated complex and proteins involved in actin turnover by MS pull-down. Both SKO cells showed major abnormalities of cell attachment, detachment and directional migration indicative of functional alterations in cell-matrix and cell-cell adhesion. We confirmed that FA structure and function were altered by reduced co-localisation of several AAPs (including TNS1 and FBLIM1) and by TFM. BICC1 directly interacted with F-actin and FBLIM1 in pull-down assays and with FBLIM1 by RNA-IP. Finally, we found evidence of FBLIM1 mRNA by smFISH and of local FBLIM1 translation (Puro-PLA) at FA sites in wild-type cells, both of which were markedly reduced in SKO cells. Conclusion Our data reveal the key roles for actin dynamics and local translation as novel mechanisms regulated by PC1 and BICC1 in the pathogenesis of ADPKD. We demonstrate that BICC1 and PC1 are co-regulated, forming a PC1-BICC1 complex critical for maintaining the structural integrity and function of FAs in normal kidney epithelial cells. Loss of this interaction results in major alterations in cell adhesion, migration and stress fibre organisation, features typical of the ADPKD cellular phenotype. BICC1 plays a dual role in this process via protein-protein interactions with ITGB1, F-actin and other AAPs, and through protein-RNA interactions regulating the local translation of key AAPs (FBLIM1) at FA sites.

#1514 Elevated FGF23 increases systolic, and diastolic blood pressure, and pulse pressure in non-dialysis CKD patients

Abstract Background and Aims Arterial stiffness is associated with high blood pressure. Elevated fibroblast growth factor 23 (FGF23) has been shown to produce changes in the phenotype of vascular smooth muscle cells that may cause an increase in arterial stiffness and an impairment of vascular function. Thus, we aimed to evaluate whether, in g5 non-dialysis CKD patients, higher concentration FGF23 is associated with elevated systolic (SBP), diastolic blood pressure (DBP), and pulse pressure (PP). Method We included 159 g5 non-dialysis CKD patients. Generalized additive models analyzed the association between intact FGF23, SBP, DBP, and PP. The body composition measurement was used to evaluate overhydration (OH). Given the potential non-linear relationship between iFGF23, with the other variables, iFGF23 was categorized into tertiles (T1 ≤ 389 pg/ml, T2 = 389—517 pg/ml, and T3 = >517 pg/ml). Results The mean age was 65.2 ± 14.7 years. 46% (n = 73) were female. The mean values of SBP and DBP were 158.8 ± 21.3 mmHg, and 87.2 ± 12.3 mmHg respectively, and the PP was 76.6 ± 20 mmHg. The median concentration of iFGF23 was 468.3 (268.8—904.9) pg/ml. It was observed a positive correlation between iFGF23 tertiles and SBP (p < 0.001), DBP (<0.01), PP (p = 0.02), and OH (p < 0.01) (Fig. 1). Multivariate generalized additive models showed an independent association of iFGF23 with SBP (Beta 5.16, CI 34-68), DBP (Beta 38.7, CI 24-53), and PP (Beta 36.3, CI 22.2-50.4). This association was not influenced by OH (%). Residual diuresis did not attenuate the association of iFGF23 with SBP, DBP, and PP. Conclusion Elevated FGF23 was associated with higher SBP, DBP, and PP in g5 non-dialysis CKD patients. This association was not modified by OH and residual diuresis.