scholarly journals ELTD1 Activation Induces an Endothelial-EMT Transition to a Myofibroblast Phenotype

2021 ◽  
Author(s):  
Helen Sheldon ◽  
John Alexander ◽  
Esther M. Bridges ◽  
Lucia Moreira ◽  
Svetlana Reilly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Wound Repair ◽  
Network Formation ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Immune Response Gene ◽  
Cell Contact

ELTD1 is expressed in endothelial and vascular smooth muscle cells and has a role in angiogenesis. It has been classified as an adhesion GPCR, but as yet, no ligand has been identified and its function remains unknown. To establish its role, ELTD1 was overexpressed in endothelial cells. Expression and consequently ligand independent activation of ELTD1 results in EndMT with a loss of cell-cell contact, formation of stress fibres and mature focal adhesions and an increased expression of smooth muscle actin. The effect was pro-angiogenic, increasing Matrigel network formation and endothelial sprouting. RNA-Seq analysis after the cells had undergone EndMT revealed large increases in chemokines and cytokines involved in regulating immune response. Gene set enrichment analysis of the data identified a number of pathways involved in myofibroblast biology suggesting that the endothelial cells had undergone a type II EMT. This type of EMT is involved in wound repair and is closely associated with inflammation implicating ELTD1 in these processes.

scholarly journals ELTD1 Activation Induces an Endothelial-EMT Transition to a Myofibroblast Phenotype

2021 ◽  
Vol 22 (20) ◽  
pp. 11293
Author(s):  
Helen Sheldon ◽  
John Alexander ◽  
Esther Bridges ◽  
Lucia Moreira ◽  
Svetlana Reilly ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Wound Repair ◽  
Network Formation ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Immune Response Gene ◽  
Cell Contact

ELTD1 is expressed in endothelial and vascular smooth muscle cells and has a role in angiogenesis. It has been classified as an adhesion GPCR, but as yet, no ligand has been identified and its function remains unknown. To establish its role, ELTD1 was overexpressed in endothelial cells. Expression and consequently ligand independent activation of ELTD1 results in endothelial-mesenchymal transistion (EndMT) with a loss of cell-cell contact, formation of stress fibres and mature focal adhesions and an increased expression of smooth muscle actin. The effect was pro-angiogenic, increasing Matrigel network formation and endothelial sprouting. RNA-Seq analysis after the cells had undergone EndMT revealed large increases in chemokines and cytokines involved in regulating immune response. Gene set enrichment analysis of the data identified a number of pathways involved in myofibroblast biology suggesting that the endothelial cells had undergone a type II EMT. This type of EMT is involved in wound repair and is closely associated with inflammation implicating ELTD1 in these processes.

scholarly journals A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts.

1996 ◽  
Vol 134 (1) ◽  
pp. 67-80 ◽  
Author(s):  
L Rønnov-Jessen ◽  
O W Petersen
Keyword(s):  
Smooth Muscle ◽  
Wound Repair ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Fibroblast Cell ◽  
Time Lapse ◽  
Alpha Smooth Muscle Actin ◽  
Two Dimensional Gel Electrophoresis ◽  
Knock Out ◽  
Migration Assay

Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-Sm actin has been found also to be expressed transiently in certain nonmuscle cells, in particular fibroblasts, which are referred to as myofibroblasts. The functional significance of alpha-sm actin in fibroblasts is unknown. However, myofibroblasts appear to play a prominent role in stromal reaction in breast cancer, at the site of wound repair, and in fibrotic reactions. Here, we show that the presence of alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of alpha-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against beta-actin. Time-lapse video microscopy revealed migratory rates of 4.8 and 3.0 microns/h, respectively. To knock out the alpha-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UTI, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of alpha-sm actin mRNA, was found to block alpha-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UTI significantly increased motility compared with the corresponding sense ODN. alpha-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

Age-Dependent Transcriptional Alterations in Cardiac Endothelial Cells

2021 ◽  
Author(s):  
Uchenna Emechebe ◽  
Jonathan William Nelson ◽  
Nabil J. Alkayed ◽  
Sanjiv Kaul ◽  
Andrew C Adey ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Single Cell ◽  
Significant Risk ◽  
Enrichment Analysis ◽  
Gene Set Enrichment Analysis ◽  
Mouse Heart ◽  
Age Related ◽  
Organ Systems ◽  
Transcriptional Alterations

Aging is a significant risk factor for cardiovascular disease. Despite the fact that endothelial cells play critical roles in cardiovascular function and disease, the molecular impact of aging on this cell population in many organ systems remains unknown. In this study, we sought to determine age-associated transcriptional alterations in cardiac endothelial cells. Highly enriched populations of endothelial cells (ECs) isolated from the heart, brain and kidney of young (3 months) and aged (24 months) C57/BL6 mice were profiled for RNA expression via bulk RNA sequencing. Approximately 700 cardiac endothelial transcripts significantly differ by age. Gene set enrichment analysis indicated similar patterns for cellular pathway perturbations. Receptor-ligand comparisons indicated parallel alterations in age-affected circulating factors and cardiac endothelial-expressed receptors. Single-cell RNA-seq analysis identified 9 distinct endothelial cell subtypes in the heart with an age-associated population shift observed for the Aplnr-enriched endothelial cell clusters. Gene and pathway enrichment analyses show that age-related transcriptional response of cardiac endothelial cells is distinct from that of endothelial cells derived from the brain or kidney vascular bed. Furthermore, single-cell analysis identified 9 distinct EC subtypes, and shows that the Aplnr-enriched subtype is reduced with age in mouse heart. Finally, we identify age-dysregulated genes in specific aged cardiac endothelial subtypes.

scholarly journals Circular RNA circ_0003204 inhibits proliferation, migration and tube formation of endothelial cell in atherosclerosis via miR-370-3p/TGFβR2/phosph-SMAD3 axis

2020 ◽  
Vol 27 (1) ◽  
Author(s):  
Shanchao Zhang ◽  
Guixiang Song ◽  
Jing Yuan ◽  
Shan Qiao ◽  
Shan Xu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ectopic Expression ◽  
Enrichment Analysis ◽  
Tube Formation ◽  
Gene Set Enrichment Analysis ◽  
Luciferase Assay ◽  
Circular Rnas ◽  
Human Aorta ◽  
Cerebral Atherosclerosis ◽  
Potential Biomarker

Abstract Background Circular RNAs (circRNAs) represent a class of non-coding RNAs (ncRNAs) which are widely expressed in mammals and tissue-specific, of which some could act as critical regulators in the atherogenesis of cerebrovascular disease. However, the underlying mechanisms by which circRNA regulates the ectopic phenotype of endothelial cells (ECs) in atherosclerosis remain largely elusive. Methods CCK-8, transwell, wound healing and Matrigel assays were used to assess cell viability, migration and tube formation. QRT-qPCR and Immunoblotting were used to examine targeted gene expression in different groups. The binding sites of miR-370-3p (miR-370) with TGFβR2 or hsa_circ_0003204 (circ_0003204) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and RNA immunoprecipitation (RIP) assay. The localization of circ_0003204 and miR-370 in ECs were investigated by fluorescence in situ hybridization (FISH). Gene function and pathways were enriched through Metascape and gene set enrichment analysis (GSEA). The association of circ_0003204 and miR-370 in extracellular vesicles (EVs) with clinical characteristics of patients were investigated using multiple statistical analysis. Results Circ_0003204, mainly located in the cytoplasm of human aorta endothelial cells (HAECs), was upregulated in the ox-LDL-induced HAECs. Functionally, the ectopic expression of circ_0003204 inhibited proliferation, migration and tube formation of HAECs exposed to ox-LDL. Mechanically, circ_0003204 could promote protein expression of TGFβR2 and its downstream phosph-SMAD3 through sponging miR-370, and miR-370 targeted the 3′ untranslated region (UTR) of TGFβR2. Furthermore, the expression of circ_0003204 in plasma EVs was upregulated in the patients with cerebral atherosclerosis, and represented a potential biomarker for diangnosis and prognosis of cerebrovascular atherogenesis. Conclusions Circ_0003204 could act as a novel stimulator for ectopic endothelial inactivation in atherosclerosis and a potential biomarker for cerebral atherosclerosis.

scholarly journals Identification of Key Candidate Genes and Pathways of Candida albicans-Infected Human Umbilical Vein Endothelial Cells and Drug Screening

2019 ◽  
Vol 60 (1) ◽  
pp. 62-69
Author(s):  
Wei Jiang ◽  
Ping Liu ◽  
Jianlei Zhang ◽  
Wenjie Yang
Keyword(s):  
Endothelial Cells ◽  
Candida Albicans ◽  
Signaling Pathways ◽  
Umbilical Vein ◽  
Enrichment Analysis ◽  
Cell Receptor ◽  
Gene Set Enrichment Analysis ◽  
Purine Nucleotide ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

AbstractCandida albicans is a common opportunistic pathogen that can cause serious infection by blood transmission. C. albicans enters the blood circulation and adheres to the endothelial cells of the vascular wall. However, the detailed mechanism of the effect of C. albicans on the endothelial cells remains unclear. In this study, the microarray expression profile of human umbilical vein endothelial cells exposed to C. albicans was analyzed. The 191 up-regulated genes were enriched in TNF, T cell receptor, and NF-kappa B signaling pathways. The 71 down-regulated genes were enriched in pyruvate metabolic, purine nucleotide metabolic, purine nucleotide biosynthetic, and humoral immune response processes. Gene set enrichment analysis showed that apoptosis, oxidative phosphorylation, IL6/JAK/STAT3 signaling pathways were enriched. Moreover, two hub genes with a high degree of connectivity, namely, MYC and IL6, were selected. Molecular screening of traditional Chinese medicine libraries was performed on the basis of the structure of MYC protein. The okanin had the highest docking score. MYC might be used as molecular targets for treatment. In addition, okanin may inhibit the infection of C. albicans. Thus, MYC can be subjected to further research.

scholarly journals α-Smooth Muscle Actin Is Crucial for Focal Adhesion Maturation in Myofibroblasts

2003 ◽  
Vol 14 (6) ◽  
pp. 2508-2519 ◽  
Author(s):  
Boris Hinz ◽  
Vera Dugina ◽  
Christoph Ballestrem ◽  
Bernhard Wehrle-Haller ◽  
Christine Chaponnier
Keyword(s):  
Smooth Muscle ◽  
Contractile Activity ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Serum Levels ◽  
Stress Fibers ◽  
Muscle Actin ◽  
Cytoplasmic Actin ◽  
Β3 Integrin ◽  
Two Phases

Cultured myofibroblasts are characterized by stress fibers, containing α-smooth muscle actin (α-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of α-SMA–negative fibroblasts. We have investigated the role of α-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of α-SMA–negative fibroblasts. Then, myofibroblasts incorporate α-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When α-SMA expression is induced in 5 d culture by TGFβ or low serum levels, fibroblast adhesion is further increased correlating with a “supermaturation” of FAs. Treatment of myofibroblasts with α-SMA fusion peptide (SMA-FP), which inhibits α-SMA–mediated contractile activity, reduces their adhesion to the level of α-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and β3 integrin-containing FAs; α5 integrin distribution is not affected. FRAP of β3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high α-SMA–mediated contractile activity of myofibroblast stress fibers.

scholarly journals Smad2 and Smad3 Play Different Roles in Rat Hepatic Stellate Cell Function and α-Smooth Muscle Actin Organization

2005 ◽  
Vol 16 (9) ◽  
pp. 4214-4224 ◽  
Author(s):  
Masayuki Uemura ◽  
E. Scott Swenson ◽  
Marianna D.A. Gaça ◽  
Frank J. Giordano ◽  
Michael Reiss ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Transforming Growth Factor ◽  
Cell Function ◽  
Stellate Cell ◽  
Smooth Muscle Actin ◽  
Focal Adhesions ◽  
Transforming Growth Factor Β ◽  
Dominant Negative ◽  
Muscle Actin ◽  
Actin Organization

Hepatic stellate cells (HSC) play a central role in the pathogenesis of liver fibrosis, transdifferentiating in chronic liver disease from “quiescent” HSC to fibrogenic myofibroblasts. Transforming growth factor-β (TGF-β), acting both directly and indirectly, is a critical mediator of this process. To characterize the function of the TGF-β signaling intermediates Smad2 and Smad3 in HSC, we infected primary rat HSC in culture with adenoviruses expressing wild-type and dominant negative Smads 2 and 3. Smad3-overexpressing cells exhibited increased deposition of fibronectin and type 1 collagen, increased chemotaxis, and decreased proliferation compared with uninfected cells and those infected with Smad2 or either dominant negative, demonstrating different biological functions for the two Smads. Additionally, coinfection experiments suggested that Smad2 and Smad3 signal via independent pathways. Smad3-overexpressing cells as well as TGF-β-treated cells demonstrated more focal adhesions and increased α-smooth muscle actin (α-SMA) organization in stress fibers, although all cells reached the same level of α-SMA expression, indicating that Smad3 also regulates cytoskeletal organization in HSC. We suggest that TGF-β, signaling via Smad3, plays an important role in the morphological and functional maturation of hepatic myofibroblasts.

scholarly journals Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

2015 ◽  
Vol 26 (14) ◽  
pp. 2698-2711 ◽  
Author(s):  
Monica Hellesøy ◽  
James B. Lorens
Keyword(s):  
Endothelial Cells ◽  
Map Kinase ◽  
Kinase Activity ◽  
Network Formation ◽  
Endothelial Cell Migration ◽  
Cell Contact ◽  
Kinase Signaling ◽  
Map Kinase Signaling ◽  
Cellular Context ◽  
Signaling Thresholds

The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis.

scholarly journals Host-Derived Pericytes and Sca-1+ Cells Predominate in the MART-1− Stroma Fraction of Experimentally Induced Melanoma

2011 ◽  
Vol 59 (12) ◽  
pp. 1060-1075 ◽  
Author(s):  
J. Humberto Treviño-Villarreal ◽  
Douglas A. Cotanche ◽  
Rosalinda Sepúlveda ◽  
Magda E. Bortoni ◽  
Otto Manneberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Endothelial Cells ◽  
Smooth Muscle ◽  
Blood Vessel ◽  
Smooth Muscle Actin ◽  
Cell Types ◽  
Muscle Actin ◽  
Cellular Composition ◽  
Tumor Capsule ◽  
Form Blood Vessel

Identification of cell types in tumor-associated stroma that are involved in the development of melanoma is hampered by their heterogeneity. The authors used flow cytometry and immunohistochemistry to demonstrate that anti–MART-1 antibodies can discriminate between melanoma and stroma cells. They investigated the cellular composition of the MART-1−, non-hematopoietic melanoma-associated stroma, finding it consisted mainly of Sca-1+ and CD146+ cells. These cell types were also observed in the skin and muscle adjacent to developing melanomas. The Sca-1+ cell population was observed distributed in the epidermis, hair follicle bulges, and tumor capsule. The CD146+ population was found distributed within the tumor, mainly associated with blood vessels in a perivascular location. In addition to a perivascular distribution, CD146+ cells expressed α-smooth muscle actin, lacked expression of endothelial markers CD31 and CD34, and were therefore identified as pericytes. Pericytes were found to be associated with CD31+ endothelial cells; however, some pericytes were also observed associated with CD31−, MART-1+ B16 melanoma cells that appeared to form blood vessel structures. Furthermore, the authors observed extensive nuclear expression of HIF-1α in melanoma and stroma cells, suggesting hypoxia is an important factor associated with the melanoma microenvironment and vascularization. The results suggest that pericytes and Sca-1+ stroma cells are important contributors to melanoma development.

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