scholarly journals Single-cell metabolic imaging reveals a RhoA-triggered glycolytic burst in motile endothelial cells

2021 ◽  
Author(s):  
David Wu ◽  
Devin Harrison ◽  
Teodora Szasz ◽  
Chih-Fan Yeh ◽  
Tzu-Pin Shentu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Glucose Transporter ◽  
Rho Gtpase ◽  
Control Cell ◽  
Lactate Production ◽  
Endothelial Activation ◽  
Metabolic Energy ◽  
Metabolic Imaging ◽  
Cell Mobility

Abstract Single-cell motility is spatially heterogeneous and driven by metabolic energy. Direct linking cell mobility to cell metabolism is technically challenging but biologically important. Here we implemented a single-cell metabolic imaging assay to measure glycolysis in individual endothelial cells using genetically-encoded biosensors capable of deciphering metabolic heterogeneity with subcellular resolution. We observed that cellular glycolysis fuels endothelial activation, migration and contraction and that the high lactate production sites co-localize with active cytoskeletal remodeling within an endothelial cell. Mechanistically, we found RhoA induces endothelial glycolysis for the phosphorylation of cofilin and myosin light chain in order to reorganize the cytoskeleton and thus control cell mobility; RhoA activation triggers a glycolytic burst through the translocation of a glucose transporter SLC2A3/GLUT3 to fuel the cellular contractile machinery, as demonstrated across multiple endothelial types. Together, our results discovered that Rho-GTPase signaling coordinates energetic metabolism with cytoskeleton remodeling to regulate the motility of single endothelial cells.

scholarly journals Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells

2021 ◽  
Vol 3 (5) ◽  
pp. 714-727
Author(s):  
David Wu ◽  
Devin L. Harrison ◽  
Teodora Szasz ◽  
Chih-Fan Yeh ◽  
Tzu-Pin Shentu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Metabolic Imaging

scholarly journals Selectivity of Guanine Nucleotide Exchange Factor-mediated Cdc42 activation in primary human endothelial cells

2019 ◽  
Author(s):  
Nathalie R. Reinhard ◽  
Sanne van der Niet ◽  
Anna Chertkova ◽  
Marten Postma ◽  
Peter L. Hordijk ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Actin Dynamics ◽  
Pleckstrin Homology Domain ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Dh Domain

AbstractThe Rho GTPase family is involved in actin dynamics and regulates the barrier function of the endothelium. One of the main barrier-promoting Rho GTPases is Cdc42, also known as cell division control protein 42 homolog. Currently, regulation of Cdc42-based signaling networks in endothelial cells (ECs) lack molecular details. To examine these, we focused on a subset of 15 Rho guanine nucleotide exchange factors (GEFs), which are expressed in the endothelium. By performing single cell FRET measurements with Rho GTPase biosensors in primary human ECs, we monitored GEF efficiency towards Cdc42 and Rac1. A new, single cell-based analysis was developed and used to enable the quantitative comparison of cellular activities of the full-length GEFs. Our data reveal a specific GEF dependent activation profile, with most efficient Cdc42 activation induced by PLEKHG2, FGD1, PLEKHG1 and pRex1 and the highest selectivity for FGD1. Additionally, we generated truncated GEF constructs that comprise only the catalytic dbl homology (DH) domain or together with the adjacent pleckstrin homology domain (DHPH). The DH domain by itself did not activate Cdc42, whereas the DHPH domain of ITSN1, ITSN2 and PLEKHG1 showed activity towards Cdc42. Together, our study characterized endothelial GEFs that may activate Cdc42, which will be of great value for the field of vascular biology.Abstract FigureGraphical Abstract

scholarly journals Analysis of Endothelial-to-Haematopoietic Transition at the Single Cell Level identifies Cell Cycle Regulation as a Driver of Differentiation

2020 ◽  
Author(s):  
Giovanni Canu ◽  
Emmanouil Athanasiadis ◽  
Rodrigo A. Grandy ◽  
Jose Garcia-Bernardo ◽  
Paulina M. Strzelecka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Endothelial Cells ◽  
Single Cell ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Time Window ◽  
Control Cell ◽  
Quiescent State ◽  
Differentiation Potential

ABSTRACTHaematopoietic stem cells (HSC) first arise during development in the aorta-gonad-mesonephros (AGM) region of the embryo from a population of haemogenic endothelial cells which undergo endothelial-to-haematopoietic transition (EHT). Despite the progress achieved in recent years, the molecular mechanisms driving EHT are still poorly understood, especially in human where the AGM region is not easily accessible. In this study, we took advantage of a human pluripotent stem cell (hPSC) differentiation system and single-cell transcriptomics to recapitulate EHT in vitro and uncover mechanisms by which the haemogenic endothelium generates early haematopoietic cells. We show that most of the endothelial cells reside in a quiescent state and progress to the haematopoietic fate within a defined time window, within which they need to re-enter into the cell cycle. If cell cycle is blocked, haemogenic endothelial cells lose their EHT potential and adopt a non-haemogenic identity. Furthermore, we demonstrated that CDK4/6 and CDK1 play a key role not only in the transition but also in allowing haematopoietic progenitors to establish their full differentiation potential. Therefore, we propose a direct link between the molecular machineries that control cell cycle progression and EHT.

scholarly journals Single-cell lactate production rate as a measure of glycolysis in endothelial cells

2021 ◽  
Vol 2 (3) ◽  
pp. 100807
Author(s):  
Devin Harrison ◽  
David Wu ◽  
Jun Huang ◽  
Yun Fang
Keyword(s):  
Endothelial Cells ◽  
Single Cell ◽  
Production Rate ◽  
Lactate Production

In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

2017 ◽  
Vol 68 (6) ◽  
pp. 1341-1344
Author(s):  
Grigore Berea ◽  
Gheorghe Gh. Balan ◽  
Vasile Sandru ◽  
Paul Dan Sirbu
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Single Cell ◽  
Cell Line ◽  
Bone Repair ◽  
Umbilical Vein ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

scholarly journals Single-Cell Transcriptional Heterogeneity of Lymphatic Endothelial Cells in Normal and Inflamed Murine Lymph Nodes

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1371
Author(s):  
Eliane Sibler ◽  
Yuliang He ◽  
Luca Ducoli ◽  
Nadja Keller ◽  
Noriki Fujimoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Lymph Nodes ◽  
Single Cell ◽  
In Silico Analysis ◽  
Skin Inflammation ◽  
Lymphatic Endothelial Cells ◽  
Depth Analysis ◽  
Subcapsular Sinus ◽  
Transcriptional Changes ◽  
Sinus Floor

The lymphatic system plays a crucial role in immunity and lymph nodes (LNs) undergo drastic remodeling during inflammation. Here, we used single-cell RNA sequencing to investigate transcriptional changes in lymphatic endothelial cells (LECs) in LNs draining naïve and inflamed skin. We found that subsets of LECs lining the different LN sinuses responded individually to skin inflammation, suggesting that they exert distinct functions under pathological conditions. Among the genes dysregulated during inflammation, we confirmed an up-regulation of CD200 in the LECs lining the subcapsular sinus floor with a possible function in immune regulation. Furthermore, by in silico analysis, we predicted numerous possible interactions of LECs with diverse immune cells in the LNs and found similarities in the transcriptional changes of LN LECs in different skin inflammation settings. In summary, we provide an in-depth analysis of the transcriptional landscape of LN LECs in the naïve state and in skin inflammation.

scholarly journals Matrix Stiffness Modulates Metabolic Interaction between Human Stromal and Breast Cancer Cells to Stimulate Epithelial Motility

Metabolites ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 432
Author(s):  
Iván Ponce ◽  
Nelson Garrido ◽  
Nicolás Tobar ◽  
Francisco Melo ◽  
Patricio C. Smith ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Stromal Cells ◽  
Glucose Transporter ◽  
Lactate Production ◽  
Resonance Energy ◽  
Metabolic Reprogramming ◽  
Dependent Manner ◽  
Functional Link

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

scholarly journals Metabolic Anti-Cancer Effects of Melatonin: Clinically Relevant Prospects

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 3018
Author(s):  
Marek Samec ◽  
Alena Liskova ◽  
Lenka Koklesova ◽  
Kevin Zhai ◽  
Elizabeth Varghese ◽  
...  
Keyword(s):  
Cancer Metabolism ◽  
Glucose Transporter ◽  
Aerobic Glycolysis ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Pentose Phosphate ◽  
Metabolic Reprogramming ◽  
Effective Prevention ◽  
Anti Cancer ◽  
Glucose 6 Phosphate Dehydrogenase

Metabolic reprogramming characterized by alterations in nutrient uptake and critical molecular pathways associated with cancer cell metabolism represents a fundamental process of malignant transformation. Melatonin (N-acetyl-5-methoxytryptamine) is a hormone secreted by the pineal gland. Melatonin primarily regulates circadian rhythms but also exerts anti-inflammatory, anti-depressant, antioxidant and anti-tumor activities. Concerning cancer metabolism, melatonin displays significant anticancer effects via the regulation of key components of aerobic glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP) and lipid metabolism. Melatonin treatment affects glucose transporter (GLUT) expression, glucose-6-phosphate dehydrogenase (G6PDH) activity, lactate production and other metabolic contributors. Moreover, melatonin modulates critical players in cancer development, such as HIF-1 and p53. Taken together, melatonin has notable anti-cancer effects at malignancy initiation, progression and metastasing. Further investigations of melatonin impacts relevant for cancer metabolism are expected to create innovative approaches supportive for the effective prevention and targeted therapy of cancers.

scholarly journals In Vivo Assessment of Metabolic Abnormality in Alport Syndrome Using Hyperpolarized [1-13C] Pyruvate MR Spectroscopic Imaging

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 222
Author(s):  
Nguyen-Trong Nguyen ◽  
Eun-Hui Bae ◽  
Luu-Ngoc Do ◽  
Tien-Anh Nguyen ◽  
Ilwoo Park ◽  
...  
Keyword(s):  
Disease Progression ◽  
Alport Syndrome ◽  
Genetic Disorder ◽  
Lactate Production ◽  
Clinical Importance ◽  
Metabolic Imaging ◽  
Renal Metabolism ◽  
Hyperpolarized 13C ◽  
And Control

Alport Syndrome (AS) is a genetic disorder characterized by impaired kidney function. The development of a noninvasive tool for early diagnosis and monitoring of renal function during disease progression is of clinical importance. Hyperpolarized 13C MRI is an emerging technique that enables non-invasive, real-time measurement of in vivo metabolism. This study aimed to investigate the feasibility of using this technique for assessing changes in renal metabolism in the mouse model of AS. Mice with AS demonstrated a significant reduction in the level of lactate from 4- to 7-week-old, while the levels of lactate were unchanged in the control mice over time. This reduction in lactate production in the AS group accompanied a significant increase of PEPCK expression levels, indicating that the disease progression in AS triggered the gluconeogenic pathway and might have resulted in a decreased lactate pool size and a subsequent reduction in pyruvate-to-lactate conversion. Additional metabolic imaging parameters, including the level of lactate and pyruvate, were found to be different between the AS and control groups. These preliminary results suggest that hyperpolarized 13C MRI might provide a potential noninvasive tool for the characterization of disease progression in AS.

Sign in / Sign up

Export Citation Format

Share Document