Lymphocyte-Activating Chemokines Are Potent Inducers of Sudden Arrest and Firm Adhesion in Mesenchymal Stromal Cells (MSCs) Under Shear Flow

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2425-2425
Author(s):  
Felicia Ciuculescu ◽  
Melanie Giesen ◽  
Erika Deak ◽  
Erhard Seifried ◽  
Tanja Nicole Hartmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Parallel Plate ◽  
Flow Cytometric Analysis ◽  
Transendothelial Migration ◽  
Intravenous Route ◽  
Pre Treatment

Abstract Mesenchymal Stromal Cells (MSCs) are being developed as a cellular therapeutic, and used clinically used for induction of immunomodulation and suppression of inflammation after application via the intravenous route. However, relatively little is known on how MSCs interact with the vessel wall to induce tissue-specific egress. To elucidate potentially underlying mechanisms, we analyzed human bone-marrow derived MSCs in parallel plate flow chambers and characterized their interaction with endothelial cells or immobilized endothelial ligands as to tethering and rolling, sudden arrest, adhesion strengthening, and transendothelial migration. Flow cytometric analysis of MSCs confirmed the known expression predominantly of integrins α4, α5 and β1, and revealed only low detectable levels of a range of chemokine receptors including CCR1, 5, 6, 7 and CXCR1, 4 and 5. To investigate a potential role of chemokines in the adhesion process of MSCs, we first seeded MSCs without shear onto immobilized recombinant (r) VCAM-Fc fusion protein without or with co-immobilized chemokines in parallel plate flow chambers for 3 min, and subsequently started flow by stepwise increasing shear stress from 0.35 to 15 dynes/cmE2. Co-immobilization of various chemokines including CCL15/HCC-2, a known activator of CCR1 and 3, CCL20/LARC (of CCR6), CCL19/ELC (of CCR7), CXCL8/IL-8 (of CXCR1/2), CXCL12/SDF-1 (of CXCR4) and CXCL13/BCA-1 (of CXCR5) with rVCAM-1 increased shear-resistant binding of MSCs by up to four-fold compared to controls on rVCAM alone, with only moderate increases through CXCL8/IL-8 and CXCL12/SDF-1 and strongest effects through CCL19/ELC and CCL20/LARC. The reverse was observed in hematopoetic progenitor cells, which responded best to CXCL12/SDF-1. No or little adhesion of MSCs to rICAM-1 was observed under analogous conditions. We next chose to study CXCL12/SDF-1 and CCL19/ELC in more detail on intact endothelial cells. MSCs were flushed through parallel plate flow chambers on Human Umbilical Vein Endothelial Cells (HUVECs) at an initial shear stress of 0.35 or 0.5 dyn/cmE2 for 1 min followed by an elevated shear stress of 5 dyn/cmE2 for further 10 min. We found that rolling of MSCs was increased after pre-treatment of HUVECs with TNF-α but only few spontaneous arrests were obsereved under these conditions. However, when HUVECs were additionally overlaid with CXCL12/SDF-1 or CCL19/ELC, MSCs arrested firmly on the endothelium. Pretreatment of MSCs with function-blocking anti-CD44 or anti-VLA-4 antibodies, or pre-treatment of HUVECs with anti-E-selectin or anti-VCAM-1 antibodies partially (anti-CD44, -E-selectin) or completely (anti-VLA-4, -VCAM-1) suppressed arrest of MSCs. Analysis of adhesion strengthening on HUVECs as well as on immobilized rVCAM-1 confirmed the mediation of shear-resistant binding of MSCs by CXCL12/SDF-1 and CCL19/ELC. Pre-incubation of MSCs with pertussis toxin also suppressed both, sudden arrest and firm adhesion, indicating a role of signalling through G protein coupled receptor via Gαi. Finally, MSCs were able to transmigrate chemokine-coated HUVECs but not untreated. We conclude that MSCs respond to individual chemokines with different efficiencies to induce arrest and firm adhesion before their transendothelial migration. MSCs were found highly responsive to chemokines which physiologically act on lymphocytes to enter secondary lymphoid organs or sites of inflammation. This points to a preference of MSCs to follow lymphocyte egress pathways and reach sites of inflammation to induce immunomodulation and –suppression.

Mesenchymal Stromal Cells (MSCs) Lack Adhesion to Endothelial Cells through Selectins and b2 Integrins and Interact Dominantly Using Integrin a4b1 Activated by GTPase Rap1.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2589-2589
Author(s):  
Olga Nilmaer ◽  
Melanie Giesen ◽  
Erika Deak ◽  
Victoria Lang ◽  
Johannes Leibacher ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Conflicts Of Interest ◽  
Shear Stresses ◽  
Lower Efficiency ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Integrin Ligand

Abstract Abstract 2589 Mesenchymal Stromal Cells (MSCs) are increasingly used in patients e.g. to combat Graft-versus-Host-Disease or to support hematopoietic regeneration after allogeneic Hematopoietic Stem Cell Transplantation. However, both the dynamics and the mechanisms by which intravenously transplanted MSCs (n=5 donors) interact with the vessel wall are incompletely characterized. We compared the ability of MSCs and blood leukocytes (PBMCs; n=5) to interact with known endothelial ligands using a flow chamber system and transplantation into immunodeficient mice. Although recombinant P- and E-selectin reduced the flow speed of MSCs significantly by 30±3% (means ± SD), which was reversible in the presence of function-blocking anti P- or E-selectin antibodies MSCs were induced to roll at speeds of 1–5 μm/s at much lower efficiency as PBMCs at shear stresses of 0.35 – 8 dyn/cm2 (MSCs, 2±1%; PBMCs, 21±4% of interacting cells; means ± SD). In contrast, immobilized recombinant VCAM-1 Ig under these conditions dose-dependently induced interactions of MSCs with a comparable efficiency as of PBMCs, leading to stable arrest. Whereas 33 ± 8% (means ± SD) of PBMCs rolled on 20 μg/ml VCAM-1 Ig, MSCs howerver lacked rolling behaviour but always directly stopped. Analysis of the velocity vectors of individual MSCs revealed an staggering interaction of MSCs during the stopping process with prolonged distances until they reached stable arrest (58±10 vs 14±3 μm for MSCs and PBMCs, respectively (means ± SD, p=0.004). Whereas PBMCs were induced to both roll and arrest on the b2 integrin ligand ICAM-1 and on the a4b7 integrin ligand MAdCAM-1 at 0.35–2 dyn/cm2, MSCs were incapable to roll or arrest under these conditions. Analysis of MSC arrest behaviour on HUVEC endothelial cells pretreated with TNF-a that expressed both VCAM-1 and ICAM-1 and coated with chemokines CXCL12 or CCL19 showed that MSCs arrested with only 14±5% of the efficiency of PBMCs at 0.35dyn/cm2. Function blocking antibodies confirmed the pivotal role of a4b1/VCAM-1 dependent adhesion in stable arrest of MSCs. We found that MSCs bound to the HUVECs more rigidly than PBMCs since, when arrested cells were exposed to increased shear stress of 5 dyn/cm2 only 3±4% of MSCs de-adhered, in contrast to PBMCs which 74±4% de-adhered. Chemokine-induced signals were instrumental in strengthening adhesion of MSCs since for CXCL12-induced MSCs to endothelial cells adhesion was counteracted (i) pretreatment with pertussis toxin, (ii) by application of the CXCR4 inhibitor (AMD3100) and (iii) after siRNA mediated downregulation of b arrestins 1 or 2, or the GPCR kinase (GRK)-6, which known to be involved in chemokine receptor signalling. All above treatments lead to significantly decreased numbers of MSCs that remained adherent at shear stresses between 2 and 15 dyn/cm2. Since cDNA microarray analises using MSCs exposed to shear stress for 0–12h indicated, among others, upregulation of the GTPase Rap1A and its activator, PDZ-GEF1, we analyzed activation of Rap1 proteins using a pulldown assay in a CXCL12-induced manner. MSCs depleted of Rap1A and B proteins by transfection with siRNA showed a significant reduction of stably arresting cells (>10s) on HUVEC precoated with CCL19 to 66±4% of controls (p=0.04). Moreover, siRNA mediated depletion of Rap1A and B in MSCs resulted in a >60% reduction in numbers of transplanted MSCs accumulating in the lungs of NOD/SCID mice, and concomitant significant increases of numbers of MSCs detected in blood (8.9±0.4 fold; p<0.05) and other tissues such as liver (2.2±0.8 fold, p<0.05) and spleen (2.1±0.7 fold, p<0.05). Together, MSCs display major deficits in adhesion dynamics and adhesion receptor usage compared with normal leukocytes. Modulation of the GTPase Rap1 may serve as a strategy to counteract the strong activation of b1 integrins and improve the circulation behaviour of transplanted MSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cells Activation and Infectability Amplifying the Angiogenic Process.

2021 ◽  
Author(s):  
Scutera Sara ◽  
Mitola Stefania ◽  
Sparti Rosaria ◽  
Salvi Valentina ◽  
Grillo Elisabetta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Cytokine Production ◽  
Bartonella Henselae ◽  
Antimicrobial Properties ◽  
Cat Scratch Disease ◽  
Angiogenic Potential ◽  
Bacillary Angiomatosis

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, B. henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of Bartonella , the mechanisms by which Bartonella induces EC activation are not completely clear, as well as the possible contribution of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections exerting antimicrobial properties but also acting as a shelter for pathogens. Here we delved into the role of MSCs as reservoir of Bartonella and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclear bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of EGFR, TLR2 and NOD1, proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors VEGF, FGF-7, MMP-9, PIGF, serpin E1, TSP-1, uPA, IL-6, PDGF-D, CCL5 and CXCL8. Supernatants from B. henselae -infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion and reorganization in tube-like structures. Altogether, these results candidate MSCs as a still underestimated niche for B. henselae persistent infection and reveal a MSC-EC crosstalk that may contribute to exacerbate bacterial-induced angiogenesis and granuloma formation.

The role of children's bone marrow mesenchymal stromal cells in the ex vivo expansion of autologous and allogeneic hematopoietic stem cells

2015 ◽  
Vol 39 (10) ◽  
pp. 1099-1110 ◽  
Author(s):  
Iordanis Pelagiadis ◽  
Eftichia Stiakaki ◽  
Christianna Choulaki ◽  
Maria Kalmanti ◽  
Helen Dimitriou
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem

scholarly journals Shear stress augments mitochondrial ATP generation that triggers ATP release and Ca2+ signaling in vascular endothelial cells

2018 ◽  
Vol 315 (5) ◽  
pp. H1477-H1485 ◽  
Author(s):  
Kimiko Yamamoto ◽  
Hiromi Imamura ◽  
Joji Ando
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular Endothelial Cells ◽  
Critical Role ◽  
Atp Release ◽  
Vascular Endothelial ◽  
The Novel ◽  
Caveolin 1 ◽  
Atp Generation

Vascular endothelial cells (ECs) sense and transduce hemodynamic shear stress into intracellular biochemical signals, and Ca2+ signaling plays a critical role in this mechanotransduction, i.e., ECs release ATP in the caveolae in response to shear stress and, in turn, the released ATP activates P2 purinoceptors, which results in an influx into the cells of extracellular Ca2+. However, the mechanism by which the shear stress evokes ATP release remains unclear. Here, we demonstrated that cellular mitochondria play a critical role in this process. Cultured human pulmonary artery ECs were exposed to controlled levels of shear stress in a flow-loading device, and changes in the mitochondrial ATP levels were examined by real-time imaging using a fluorescence resonance energy transfer-based ATP biosensor. Immediately upon exposure of the cells to flow, mitochondrial ATP levels increased, which was both reversible and dependent on the intensity of shear stress. Inhibitors of the mitochondrial electron transport chain and ATP synthase as well as knockdown of caveolin-1, a major structural protein of the caveolae, abolished the shear stress-induced mitochondrial ATP generation, resulting in the loss of ATP release and influx of Ca2+ into the cells. These results suggest the novel role of mitochondria in transducing shear stress into ATP generation: ATP generation leads to ATP release in the caveolae, triggering purinergic Ca2+ signaling. Thus, exposure of ECs to shear stress seems to activate mitochondrial ATP generation through caveola- or caveolin-1-mediated mechanisms. NEW & NOTEWORTHY The mechanism of how vascular endothelial cells sense shear stress generated by blood flow and transduce it into functional responses remains unclear. Real-time imaging of mitochondrial ATP demonstrated the novel role of endothelial mitochondria as mechanosignaling organelles that are able to transduce shear stress into ATP generation, triggering ATP release and purinoceptor-mediated Ca2+ signaling within the cells.

PPARβ/δ Priming Enhances the Anti-Apoptotic and Therapeutic Properties of Mesenchymal Stromal Cells in Myocardial Ischemia-Reperfusion Injury

2021 ◽  
Author(s):  
Charlotte Sarre ◽  
Rafael Contreras Lopez ◽  
Nitirut Nerpernpisooth ◽  
Christian Barrere ◽  
Sarah Bahraoui ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Myocardial Infarction ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Phase Iii ◽  
Therapeutic Properties ◽  
Cardioprotective Effects

Abstract Background: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARβ/δ (Peroxisome proliferator-activated receptors β/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARβ/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARβ/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. Objectives: The aim of this study was to investigate the role of PPARβ/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction.Methods and results: Naïve MSC and MSC pharmacologically activated or inhibited for PPARβ/δ were challenged with H202. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARβ/δ agonist GW0742 versus naïve MSC. In addition, PPARβ/δ-priming allowed to reveal the anti-apoptotic effect of MSC on co-cultured cardiomyocytes. When injected during reperfusion in an ex vivo heart model of myocardial infarction, PPARβ/δ-primed MSC at a dose of 3.75x105 MSC/heart provided the same cardioprotective efficiency than 7.5x105 naïve MSC, identified as the optimal dose in our model. These enhanced short-term cardioprotective effects were associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 hour of reperfusion. By contrast, inhibition of PPARβ/δ before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. Conclusion: Altogether these results revealed that PPARβ/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARβ/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Abstract WP88: In a Pro-inflammatory Environment, Mesenchymal Stromal Cells Exert Anti-inflammatory and Pro-angiogenic Effects on Endothelial Cells

Stroke ◽  
2018 ◽  
Vol 49 (Suppl_1) ◽  
Author(s):  
Kaavya Giridhar ◽  
Nikunj Satani ◽  
Bing Yang ◽  
Songmi Lee ◽  
Xiaopei Xi ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Anti Inflammatory

Role of cadherins and plakoglobin in interendothelial adhesion under resting conditions and shear stress

1997 ◽  
Vol 273 (5) ◽  
pp. H2396-H2405 ◽  
Author(s):  
Hans-Joachim Schnittler ◽  
Bernd Püschel ◽  
Detlev Drenckhahn
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Time Course ◽  
Fluid Shear Stress ◽  
Vascular Endothelial ◽  
Fluid Shear ◽  
Platelet Endothelial Cell Adhesion ◽  
Cell Adhesion Molecule 1 ◽  
Arterial Endothelial Cells

The role of cadherins and the cadherin-binding cytosolic protein plakoglobin in intercellular adhesion was studied in cultured human umbilical venous endothelial cells exposed to fluid shear stress. Extracellular Ca2+depletion (<10−7 M) caused the disappearance of both cadherins and plakoglobin from junctions, whereas the distribution of platelet endothelial cell adhesion molecule 1 (PECAM-1) remained unchanged. Cells stayed fully attached to each other for several hours in low Ca2+ but began to dissociate under flow conditions. At the time of recalcification, vascular endothelial (VE) cadherin and β-catenin became first visible at junctions, followed by plakoglobin with a delay of ∼20 min. Full fluid shear stress stability of the junctions correlated with the time course of the reappearance of plakoglobin. Inhibition of plakoglobin expression by microinjection of antisense oligonucleotides did not interfere with the junctional association of VE-cadherin, PECAM-1, and β-catenin. The plakoglobin-deficient cells remained fully attached to each other under resting conditions but began to dissociate in response to flow. Shear stress-induced junctional dissociation was also observed in cultures of plakoglobin-depleted arterial endothelial cells of the porcine pulmonary trunk. These observations show that interendothelial adhesion under hydrodynamic but not resting conditions requires the junctional location of cadherins associated with plakoglobin. β-Catenin cannot functionally compensate for the junctional loss of plakoglobin, and PECAM-1-mediated adhesion is not sufficient for monolayer integrity under flow.

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