Nayden Naydenov - Academia.edu (original) (raw)

Papers by Nayden Naydenov

The FASEB Journal

Integrity of the intestinal epithelial barrier is regulated by two types of adhesive structures: ... more Integrity of the intestinal epithelial barrier is regulated by two types of adhesive structures: intercellular junctions and focal adhesions (FA) that control epithelial cell attachment to each other and to the extracellular matrix (ECM), respectively. Disassembly of adherens junctions (AJ) and tight junctions (TJ) plays key roles in the breakdown of the intestinal epithelial barrier during mucosal inflammation. Abnormal architecture and functions of FA significantly contribute to attenuated restitution of the inflamed intestinal mucosa. Proinflammatory cytokines such as interferon (IFN)‐γ and tumor necrosis factor (TNF)‐α, are known to trigger AJ/TJ disassembly and attenuate wound healing in model intestinal epithelial cell monolayers. We hypothesized that inhibition of protein translation factors eIF4G1 and eIF4G2 could mediate cytokine‐induced disruption of the gut barrier and attenuation of epithelial restitution.Immunoblotting analysis of IFNγ and IFNγ/TNFα‐treated HT29 and T84...

Biochemical and Biophysical Research Communications, May 1, 2017

A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional... more A soluble N-ethylmaleimide-sensitive factor-attachment protein alpha (αSNAP) is a multifunctional scaffolding protein that regulates intracellular vesicle trafficking and signaling. In cultured intestinal epithelial cells, αSNAP has been shown to be essential for cell survival, motility, and adhesion; however, its physiologic functions in the intestinal mucosa remain unknown. In the present study, we used a mouse with a spontaneous hydrocephalus with hop gait (hyh) mutation of αSNAP to examine the roles of this trafficking protein in regulating intestinal epithelial homeostasis in vivo. Homozygous hyh mice demonstrated decreased expression of αSNAP protein in the intestinal epithelium, but did not display gross abnormalities of epithelial architecture in the colon and ileum. Such αSNAP depletion attenuated differentiation of small intestinal epithelial enteroids ex vivo. Furthermore, αSNAP-deficient mutant animals displayed reduced formation of lysozyme granules in small intestinal crypts and decreased expression of lysozyme and defensins in the intestinal mucosa, which is indicative of defects in Paneth cell differentiation. By contrast, development of Goblet cells, enteroendocrine cells, and assembly of enterocyte apical junctions was not altered in hyh mutant mice. Our data revealed a novel role of αSNAP in the intestinal Paneth cell differentiation in vivo.

Inflammatory Bowel Diseases, Apr 8, 2020

BackgroundDisruption of the gut barrier is an essential mechanism of inflammatory bowel diseases ... more BackgroundDisruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro.MethodsThe study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay.ResultsE-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling.ConclusionsOur data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

Cells, Dec 29, 2019

Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, fila... more Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.

Cellular and Molecular Life Sciences, Sep 3, 2020

Remodeling of the intracellular cytoskeleton plays a key role in accelerating tumor growth and me... more Remodeling of the intracellular cytoskeleton plays a key role in accelerating tumor growth and metastasis. Targeting different cytoskeletal elements is important for existing and future anticancer therapies. Anillin is a unique scaffolding protein that interacts with major cytoskeletal structures, e.g., actin filaments, microtubules and septin polymers. A well-studied function of this scaffolding protein is the regulation of cytokinesis at the completion of cell division. Emerging evidence suggest that anillin has other important activities in non-dividing cells, including control of intercellular adhesions and cell motility. Anillin is markedly overexpressed in different solid cancers and its high expression is commonly associated with poor prognosis of patient survival. This review article summarizes rapidly accumulating evidence that implicates anillin in the regulation of tumor growth and metastasis. We focus on molecular and cellular mechanisms of anillin-dependent tumorigenesis that include both canonical control of cytokinesis and novel poorly understood functions as a nuclear regulator of the transcriptional reprogramming and phenotypic plasticity of cancer cells.

Journal of Crohn's and Colitis, Jan 30, 2023

Oral presentations the time of anti-TNF withdrawal to make recommendations for clinical practice.... more Oral presentations the time of anti-TNF withdrawal to make recommendations for clinical practice. Aims: Primary: to compare the rates of clinical remission at 1 year in patients who discontinue anti-TNF treatment vs. those who continue treatment. Secondary objectives: to know the effect of anti-TNF withdrawal on relapse-free time, mucosal healing and safety; and to identify predictive factors for relapse. Methods: Prospective, quadruple-blind, multicentre, randomised, controlled trial. Patients with ulcerative colitis (UC) or Crohn's disease (CD) in clinical remission for > 6 months were randomised to maintain anti-TNF treatment [maintenance arm (MA)] or to withdraw it [withdrawal arm (WA)]. Patients who were on infliximab (IFX) received IFX 5 mg/kg or an intravenous placebo every 8 weeks, while patients on adalimumab (ADA) received subcutaneous ADA 40 mg or placebo every other week. Patients were followed-up until month 12 or up to the time of clinical relapse, whichever came first. Inclusion and exclusion criteria, trial scheme and definitions are summarized in figures 1a, 1b and 1c. Results were analysed by intention-to-treat (ITT) and by per-protocol (PP). Local investigators were blinded to faecal calprotectin (FC) and IFX and ADA trough levels. On-site monitoring was performed to assess data quality. Results: 159 patients were screened, from whom 140 were randomised and comprised the ITT cohort: 70 allocated to the MA and 70 to the WA. Fifteen patients dropped out before the end of follow-up (12 months or relapse), leaving 63 patients in the MA and 62 patients in the WA for the PP analysis. The characteristics of patients in the MA and WA were similar (figure 2). The proportions of patients who maintained clinical remission-59/70 (84%), 95% confidence interval (CI)=74-92% in the MA vs. 53/70 (76%), 95%CI=64-85% in the WA-and who remained without significant endoscopic lesions at the end of follow-up were similar between groups (figures 3a, 3b, 3c). Only the proportion of patients with FC >250 mg/g was higher in the WA at the end of follow-up (figure 3d). Maintenance of clinical remission was no different between groups (figure 4). The same percentage of patients in both groups had at least one adverse event (69%). The proportion of patients with serious adverse events was also similar between groups (4% in MA vs. 7% in WA). Conclusion: Anti-TNF withdrawal in selected IBD patients in clinical, endoscopic, and radiologic remission could be feasible without an increase in the risk of clinical relapse. Long-term follow-up of these patients is warranted.

Gastroenterology, May 1, 2021

American Journal of Pathology, 2021

Chronic inflammatory diseases cause profound alterations in tissue homeostasis, including uncheck... more Chronic inflammatory diseases cause profound alterations in tissue homeostasis, including unchecked activation of immune and nonimmune cells leading to disease complications such as aberrant tissue repair and fibrosis. Current anti-inflammatory therapies are often insufficient in preventing or reversing these complications. Remodeling of the intracellular cytoskeleton is critical for cell activation in inflamed and fibrotic tissues; however, the cytoskeleton has not been adequately explored as a therapeutic target in inflammation. Septins are GTP-binding proteins that self-assemble into higher order cytoskeletal structures. The septin cytoskeleton exhibits a number of critical cellular functions, including regulation of cell shape and polarity, cytokinesis, cell migration, vesicle trafficking, and receptor signaling. Surprisingly, little is known about the role of the septin cytoskeleton in inflammation. This article reviews emerging evidence implicating different septins in the regulation of hostpathogen interactions, immune cell functions, and tissue fibrosis. Targeting of the septin cytoskeleton as a potential future therapeutic intervention in human inflammatory and fibrotic diseases is also discussed.

Cells, Apr 27, 2022

This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY

The FASEB Journal, May 1, 2022

Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases ... more Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases (IBD), triggers significant changes in the intestinal epithelial homeostasis. These changes include leakiness of the gut barrier, formation of mucosal wounds and, in most severe cases, oncogenic transformation of colonic epithelium resulting in colitis‐associated colon cancer (CAC). Altered structure and dynamics of epithelial junctions is a hallmark of intestinal inflammation, mediating epithelial barrier injury and repair. P‐cadherin (gene name: CDH3) is an important component of adherens junctions (AJ), which is poorly expressed in normal intestinal epithelium, but could be induced in inflamed and injured mucosa. The goal of this his study was to investigate the roles of P‐cadherin in regulating intestinal inflammation, mucosal repair and CAC.P‐cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. Roles of P‐cadherin in intestinal inflammation and tumorigenesis in vivo were investigated using a mouse strain with total P‐cadherin knockout. Dextran sulfate sodium (DSS)‐induced colitis was utilized to study mucosal inflammation, whereas CAC was established using a classical azoxymetane (AOM)/DSS model. Severity of acute DSS colitis was not affected by P‐cadherin knockout, however, P‐cadherin null mice exhibited faster recovery after DSS withdrawal indicating accelerated repair of injured mucosa. No significant differences in the number and size of colonic tumors was observed in P‐cadherin null and control mice after AOM/DSS induced CAC. Consistently, CRISPR/Cas9‐mediated knockout of P‐cadherin in SK‐CO15 and HCA7 human colonic epithelial cell lines accelerated epithelial wound healing without affecting cell proliferation. The faster migration of P‐cadherin depleted cells was associated with diminished cell‐matrix adhesions and increased cell spreading. Loss of P‐cadherin resulted in activation of Src kinase and the pro‐migratory phenotype of P‐cadherin depleted cell was reversed by pharmacological inhibition of Src. Furthermore, accelerated epithelial wound healing caused by loss of P‐cadherin was prevented by Rac1 inhibition. Our findings highlight P‐cadherin as a negative regulator of intestinal epithelial would heling in vitro and mucosal repair in vivo. By contrast, this AJ protein appears to be dispensable for intestinal epithelial cell proliferation and CAC development.

The FASEB Journal, Apr 1, 2012

Intracellular trafficking is a key mechanism that regulates cell fate by participating in pro‐dea... more Intracellular trafficking is a key mechanism that regulates cell fate by participating in pro‐death or pro‐survival signaling. A soluble N‐ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP) is well‐known component of vesicle trafficking machinery. αSNAP increases cell resistance to cytotoxic stimuli, although mechanisms of its pro‐survival function remain poorly understood. In this study, we found that either siRNA‐mediated knockdown, or overexpression of dominat negative form of αSNAP induced apoptosis in several types of epithelial cells. This apoptosis was not caused by activation of the major pro‐death regulators Bax and p53 and was independent of a key αSNAP binding partner, NSF. Instead, death of αSNAP‐depleted cells was accompanied by down‐regulation of the anti‐apoptotic Bcl‐2 protein; it was mimicked by inhibition and attenuated by overexpression of Bcl‐2. αSNAP depletion resulted in impaired Endoplasmic Reticulum (ER) to Golgi trafficking and Golgi fragmentation. Furthermore, pharmacological disruption of Golgi/ER with brefeldin A and eeyarestatin or depletion of ER‐Golgi associated AAA‐ATPase p97 decreased Bcl‐2 level and triggered cell apoptosis. These results reveal a novel role for αSNAP in regulating epithelial cell survival. Supported by NIH grants DK083968 and DK084953 to AII.

The FASEB Journal, Apr 1, 2013

Integrin‐based cell‐matrix adhesions play critical roles in mediating differentiation, survival a... more Integrin‐based cell‐matrix adhesions play critical roles in mediating differentiation, survival and motility of epithelial cells. Vesicular trafficking is a key mechanism that regulates assembly of integrin‐based cell‐matrix adhesions and cell migration. A soluble N‐ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP) is a critical regulator of vesicle fusion machinery, however, its role in cell‐matrix adhesions remains unexplored. In this study, we found that siRNA‐mediated knockdown of αSNAP induced matrix detachment in several types of epithelial cells that was independent of a key αSNAP binding partner‐NSF. αSNAP silencing impaired proper glycosylation of β1‐integrin and its trafficking to the cell surface, decreased phosphorylation of FAK and Src and caused disassembly of focal adhesions. Pharmacological inhibition of FAK and Src, but not β1‐ integrin knock‐down recapitulated the effects of αSNAP depletion on cell‐matrix adhesions. Loss of αSNAP depletion resulted in Golgi fragmentation. Furthermore, pharmacological disruption of ER‐Golgi trafficking, or depletion of ER‐Golgi associated protein GBF1 induced FAK/Src dephosphorylation and triggered cell detachment. These results reveal a novel role for αSNAP in modulating β1‐integrin trafficking and matrix adhesions of epithelial cells. Supported by NIH grants DK083968 and DK084953 to AII.

The FASEB Journal, Apr 1, 2020

Breast cancer metastasis is driven by profound remodeling of the intracellular cytoskeleton that ... more Breast cancer metastasis is driven by profound remodeling of the intracellular cytoskeleton that enables efficient cell migration and invasion. Anillin is a unique scaffolding protein regulating major cytoskeletal structures, such as actin filaments, microtubules and septin polymers. It is markedly overexpressed in breast cancer and high anillin expression is associated with poor prognosis. The aim of this study was to investigate the role of anillin in breast cancer metastasis. CRISPR/Cas9 technology was used to deplete anillin in highly metastatic MDA‐MB‐231 and BT549 cells and to overexpress it in poorly invasive MCF10AneoT cells. The effects of anillin depletion and overexpression on breast cancer cell motility in vitro were examined by a combination of wound healing and Matrigel invasion assays. Assembly of the actin cytoskeleton and matrix adhesions was evaluated by immunofluorescence labeling and confocal microscopy. In vitro tumor development was monitored by soft agar grow, whereas cancer stem cells were examined using a mammoshpere formation assay and flow cytometry. The effects of anillin knockout on tumor growth and metastasis in vivo was determined by injecting control and anillin‐depleted breast cancer cells into NGS mice. The loss‐of‐function and gain‐of‐function studies demonstrated that anillin is necessary and sufficient to accelerate migration, invasion and anchorage‐independent growth of breast cancer cells in vitro. Furthermore, loss of anillin markedly attenuated primary tumor growth and metastasis of breast cancer in vivo. In breast cancer cells, anillin was localized in the nucleus, however knockout of this protein affected the cytoplasmic/cortical events, such as the organization of actin cytoskeleton and cell‐matrix adhesions. Furthermore, we observed a global transcriptional reprogramming of anillin‐depleted breast cancer cells that resulted in suppression of their stemness and induction of the mesenchymal to epithelial trans‐differentiation. Such trans‐differentiation was manifested by upregulation of basal keratins along with increased expression of E‐cadherin and P‐cadherin. Knockdown of E‐cadherin reversed attenuated migration and invasion of anillin‐deficient breast cancer cells. Our study demonstrates that anillin plays essential roles in promoting breast cancer growth and metastatic dissemination in vitro and in vivo and unravels novel functions of anillin in regulating breast cancer stemness and differentiation.

ABSTRACTBackgroundFibroblasts play a key role in stricture formation in Crohn’s disease (CD) but ... more ABSTRACTBackgroundFibroblasts play a key role in stricture formation in Crohn’s disease (CD) but understanding it’s pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation.MethodsWe performed scRNAseq of 13 fresh full thickness CD resections containing non-involved, inflamed non-strictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next generation sequencing, proteomics and animal models.ResultsOur integrat...

Frontiers in Immunology

Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increas... more Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization ...

Annals of the New York Academy of Sciences

The integrity and functions of epithelial barriers depend on the formation of adherens junctions ... more The integrity and functions of epithelial barriers depend on the formation of adherens junctions (AJs) and tight junctions (TJs). A characteristic feature of AJs and TJs is their association with the cortical cytoskeleton composed of actin filaments and nonmuscle myosin II (NM‐II) motors. Mechanical forces generated by the actomyosin cytoskeleton are essential for junctional assembly, stability, and remodeling. Epithelial cells express two different actin proteins and three NM‐II isoforms, all known to be associated with AJs and TJs. Despite their structural similarity, different actin and NM‐II isoforms have distinct biochemical properties, cellular distribution, and functions. The diversity of epithelial actins and myosin motors could be essential for the regulation of different steps of junctional formation, maturation, and disassembly. This review focuses on the roles of actin and NM‐II isoforms in controlling the integrity and barrier properties of various epithelia. We discuss...

Figure S11. Loss of anillin causes transcriptional reprogramming of breast cancer cells. RNA sequ... more Figure S11. Loss of anillin causes transcriptional reprogramming of breast cancer cells. RNA sequencing analysis was performed on mRNA samples isolated from control and anillin-depleted (sgRNA 3 and sgRNA 4) MDA-MB-231 cells. Gene Set enrichment analysis was performed on 275 differentially expressed genes common for two CRISPR sgRNA-derived cell lines. Red and blue bars depict cellular processes upregulated and downregulated in anillin-deficient cells, respectively.

Figure S6. Truncated anillin fragment stimulates primary breast tumor growth in vivo. MCF10AneoT ... more Figure S6. Truncated anillin fragment stimulates primary breast tumor growth in vivo. MCF10AneoT cells stably expressing either truncated anillin-GFP (tAnillin) or control GFP along with a luciferase construct were injected into the mammary gland of NSG mice. (A) Luciferase intensity at the ventral side of the mouse and tumor volume were measured starting at days 20 and 42 after injection of the cells, respectively, whereas volume, weight and total luciferase intensity of dissected tumors was measured ten weeks after the injection at the end of the experiment. (B) Luciferase intensity in isolated lungs, liver, ovary, and kidney, was measured by IVIS at the end of the experiment. Data is presented as mean ± SE (n = 10–11); **p