scholarly journals Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration

10.3791/53750 ◽  
2016 ◽  
Author(s):  
Michael A. Trembley ◽  
Lissette S. Velasquez ◽  
Eric M. Small
Keyword(s):  
Cell Migration ◽  
Ex Vivo

Attenuation of retinal endothelial cell migration and capillary morphogenesis in the absence of bcl-2

2008 ◽  
Vol 294 (6) ◽  
pp. C1521-C1530 ◽  
Author(s):  
Shuji Kondo ◽  
Yixin Tang ◽  
Elizabeth A. Scheef ◽  
Nader Sheibani ◽  
Christine M. Sorenson
Keyword(s):  
Cell Migration ◽  
Vascular System ◽  
Ex Vivo ◽  
Vascular Development ◽  
Critical Role ◽  
Endothelial Cell Migration ◽  
Cellular Functions ◽  
Capillary Morphogenesis ◽  
Angiogenesis Assay ◽  
Ischemic Retinopathy

Apoptosis plays a critical role during development and in the maintenance of the vascular system. B-cell leukemia lymphoma 2 (bcl-2) protects endothelial cells (EC) from apoptosis in response to a variety of stimuli. Previous work from this laboratory demonstrated attenuation of postnatal retinal vascular development and retinal neovascularization during oxygen-induced ischemic retinopathy in bcl-2-deficient (bcl-2−/−) mice. To gain further insight into the function of bcl-2 in the endothelium, we isolated retinal EC from bcl-2+/+ and bcl-2−/− mice. Retinal EC lacking bcl-2 demonstrated reduced cell migration, tenascin-C expression, and adhesion to vitronectin and fibronectin. The bcl-2−/− retinal EC also failed to undergo capillary morphogenesis in Matrigel. In addition, using an ex vivo angiogenesis assay, we observed reduced sprouting from aortic rings grown in culture from bcl-2−/− mice compared with bcl-2+/+ mice. Furthermore, reexpression of bcl-2 was sufficient to restore migration and capillary morphogenesis defects observed in bcl-2−/− retinal EC. Mechanistically, bcl-2−/− cells expressed significantly less endothelial nitric oxide synthase, an important downstream effecter of proangiogenic signaling. This may be attributed to increased oxidative stress in the absence of bcl-2. In fact, incubation of retinal EC or aortic rings from bcl-2−/− mice with the antioxidant N-acetylcysteine rescued their capillary morphogenesis and sprouting defects. Thus, bcl-2-mediated cellular functions play important roles not only in survival but also in proangiogenic phenotype of EC with a significant impact on vascular development and angiogenesis.

The effect of adoptive transfer of ex vivo activated T cells on the efficacy and tumor penetrance of intravenously-administered CD3-engaging bispecific antibody.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 30-30
Author(s):  
Patrick C. Gedeon ◽  
Carter M. Suryadevara ◽  
Bryan D. Choi ◽  
John H. Sampson
Keyword(s):  
T Cells ◽  
Cell Migration ◽  
T Cell ◽  
Adoptive Transfer ◽  
Bispecific Antibody ◽  
Ex Vivo ◽  
The Body ◽  
Whole Body ◽  
Activated T Cells ◽  
T Cell Migration

30 Background: Activated T cells are known to traffic throughout the body including past the blood-brain barrier where they perform routine immune surveillance. Whether activated T cells can be used to enhance the efficacy and delivery of intravenously-administered, immunotherapeutic antibodies has yet to be explored. Methods: To examine efficacy, T cell migration and antibody delivery in vivo, the invasive murine glioma, CT-2A-EGFRvIII, was implanted orthotopically in human CD3 transgenic mice. Cohorts of mice were given vehicle or 1x107 non-specifically activated, syngeneic T cells intravenously. Beginning the subsequent day, groups were treated with daily intravenous infusions of human-CD3-binding, tumor-lysis-inducing bispecific antibody (hEGFRvIII-CD3 bi-scFv) or control bispecific antibody. To block T cell extravasation, cohorts received natalizumab or isotype control via intraperitoneal injection every other day beginning on the day of adoptive cell transfer. T cell migration was assessed using whole body bioluminescence imaging of activated T cells transduced to express firefly luciferase. Bispecific antibody biodistribution was assessed using PET-CT imaging of iodine-124 labeled antibody. Results: Following intravenous administration, ex vivo activated T cells tracked to invasive, syngeneic, orthotopic glioma, reaching maximal levels on average four days following adoptive transfer. Administration of ex vivo activated T cells enhanced bispecific antibody efficacy causing a statistically significant increase in survival (p = 0.007) with 80% long-term survivors. Treatment with the T cell extravasation blocking molecule natalizumab abrogated the increase in efficacy to levels observed in cohorts that did not receive adoptive transfer of activated T cells (p = 0.922). Pre-administration with ex vivo activated T cells produced a statistically significant increase in tumor penetrance of radiolabeled bispecific antibody (p = 0.023). Conclusions: Adoptive transfer of non-specifically activated T cells enhances the efficacy and tumor penetrance of intravenously-administered CD3-binding bispecific antibody.

scholarly journals Myocardial injury after ischemia-reperfusion in mice deficient in Akt2 is associated with increased cardiac macrophage density

2011 ◽  
Vol 301 (5) ◽  
pp. H1932-H1940 ◽  
Author(s):  
Xue Li ◽  
Deana Mikhalkova ◽  
Erhe Gao ◽  
Jin Zhang ◽  
Valerie Myers ◽  
...  
Keyword(s):  
Cell Migration ◽  
Infarct Size ◽  
Actin Polymerization ◽  
Ex Vivo ◽  
Ischemia Reperfusion ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Anterior Wall ◽  
Cardiac Tissues ◽  
Macrophage Density

Akt2 protein kinase has been shown to promote cell migration and actin polymerization in several cell types, including macrophages. Because migrating macrophages constitute an important inflammatory response after myocardial ischemia, we determined cardiac macrophage expression after ischemia-reperfusion (I/R) injury and cryo-injury in mice lacking Akt2 (Akt2-KO). At 7 days post-I/R, Akt2-KO cardiac tissues showed an increase in immunohistochemical staining for macrophage markers (Galectin 3 and F4/80) compared with wild-type (WT) mice, indicating macrophage density was increased in the injured Akt2-KO myocardium. This change was time dependent because macrophage density was similar between WT and Akt2-KO myocardium at 3 days post-I/R, but by 7 and 14 days post-I/R, macrophage density was significantly increased in Akt2-KO myocardium. Concomitantly, infarct size was larger and cardiac function was reduced in Akt2-KO mice subjected to I/R. However, when cryo-infarction produced similar infarct sizes in the anterior wall in both WT and Akt2-KO mice, macrophage density remained higher in Akt2-KO mouse myocardium, suggesting Akt2 regulates myocardial macrophage density independent of infarct size. Consistently, bone marrow from Akt2-KO mice enhanced myocardial macrophage density in both C57/B6 WT and Akt2-KO recipient mice. Finally, reciprocal ex-vivo coculturing of macrophages and cardiac myocytes showed that activated Akt2-KO peritoneal macrophages had reduced mobility and adhesion when compared with WT littermate controls. Thus, although Akt-2 KO mice did not affect the initial inflammation response after injury and Akt2 deficiency has been shown to impair cell migration or motility in macrophages, our data suggested a novel mechanism in which increasing retention of Akt2-KO macrophages resulted in increasing cardiac Akt2-KO macrophage density in the myocardial space.

Ex Vivo PKH26‐Labelling of Lymphocytes for Studies of Cell Migration In Vivo

1997 ◽  
Vol 45 (5) ◽  
pp. 511-514 ◽  
Author(s):  
C. JOHNSSON ◽  
R. FESTIN ◽  
G. TUFVESON ◽  
T. H. TÖTTERMAN
Keyword(s):  
Cell Migration ◽  
Ex Vivo

scholarly journals The role of the Oncostatin M/ OSM receptor β axis in activating dermal microvascular endothelial cells in Systemic Sclerosis

2020 ◽  
Author(s):  
Grace Marden ◽  
Qianqian Wan ◽  
James Wilks ◽  
Katherine Nevin ◽  
Maria Feeney ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Ex Vivo ◽  
Oncostatin M ◽  
Microvascular Endothelial Cells ◽  
Mesenchymal Transition ◽  
Healthy Control ◽  
Microvascular Endothelial ◽  
Endothelial To Mesenchymal Transition

Abstract Background Scleroderma (SSc) is a rare autoimmune disease characterized by vascular impairment and progressive fibrosis of the skin and other organs. Oncostatin M, a member of the IL-6 family, is elevated in SSc serum and was recognized as a significant player in various stages of fibrosis. The goal of this study was to assess the contribution of the OSM/OSMRβ pathway to endothelial cell (EC) injury and activation in SSc. Methods IHC and IF were used to assess the distribution of OSM and OSMRβ in SSc (n = 14) and healthy control (n = 7) skin biopsies. Cell culture experiments were performed in human dermal microvascular endothelial cells (HDMECs) and included mRNA and protein analysis, and cell migration and proliferation assays. Ex vivo skin organoid culture was used to evaluate the effect of OSM on perivascular fibrosis. Results OSMRβ protein was elevated in dermal ECs and in fibroblasts of SSc patients. Treatments of HDMECs with OSM or IL-6 + sIL-6R have demonstrated that both cytokines similarly stimulated proinflammatory genes and genes related to endothelial-to mesenchymal transition ((EndMT). OSM was more effective than IL-6 + sIL-6R in inducing cell migration, while both treatments similarly induced cell proliferation. The effects of OSM were mediated via OSMRβ and STAT3, while the LIFR did not contribute to these responses. Both, OSM and IL-6 + sIL-6R induced profibrotic gene expression in HDMECs, as well as expansion of the perivascular PDGFRβ+ cells in the ex vivo human skin culture system. Additional studies in HDMECs showed that siRNA-mediated downregulation of FLI1 and its close homolog ERG resulted in increased expression of OSMRβ in HDMECs. Conclusions This work provides new insights into the role of the OSM/OSMRβ axis in activation/injury of dermal ECs and supports the involvement of this pathway in SSc vascular disease.

scholarly journals Short-term ex-vivo exposure to hydrogen sulfide enhances murine hematopoietic stem and progenitor cell migration, homing, and proliferation

2020 ◽  
Vol 14 (1) ◽  
pp. 214-226
Author(s):  
Anoushka Khanna ◽  
Namita Indracanti ◽  
Rina Chakrabarti ◽  
Prem Kumar Indraganti
Keyword(s):  
Cell Migration ◽  
Hydrogen Sulfide ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Short Term ◽  
Hematopoietic Stem

scholarly journals Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

10.3791/52810 ◽  
2015 ◽  
Author(s):  
Magalie Bénard ◽  
Alexis Lebon ◽  
Hitoshi Komuro ◽  
David Vaudry ◽  
Ludovic Galas
Keyword(s):  
Cell Migration ◽  
Granule Cell ◽  
Ex Vivo ◽  
Cerebellar Granule Cell ◽  
Cerebellar Granule

scholarly journals Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2

2021 ◽  
Vol 23 (1) ◽  
pp. 294
Author(s):  
Hanna Mannell ◽  
Petra Kameritsch ◽  
Heike Beck ◽  
Alexander Pfeifer ◽  
Ulrich Pohl ◽  
...  
Keyword(s):  
Cell Migration ◽  
Endothelial Cell ◽  
Connexin 43 ◽  
Ex Vivo ◽  
Tyrosine Phosphatase ◽  
Dominant Negative ◽  
Endothelial Cell Migration ◽  
Junction Protein ◽  
Endothelial Migration

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

scholarly journals C3d Elicits Neutrophil Degranulation and Decreases Endothelial Cell Migration, with Implications for Patients with Alpha-1 Antitrypsin Deficiency

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1925
Author(s):  
Laura T. Fee ◽  
Debananda Gogoi ◽  
Michael E. O’Brien ◽  
Emer McHugh ◽  
Michelle Casey ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Plasma Levels ◽  
Plasma Membranes ◽  
Ex Vivo ◽  
Endothelial Cell Migration ◽  
Increased Risk ◽  
Alpha 1 Antitrypsin ◽  
The Impact

Alpha-1 antitrypsin (AAT) deficiency (AATD) is characterized by increased risk for emphysema, chronic obstructive pulmonary disease (COPD), vasculitis, and wound-healing impairment. Neutrophils play a central role in the pathogenesis of AATD. Dysregulated complement activation in AATD results in increased plasma levels of C3d. The current study investigated the impact of C3d on circulating neutrophils. Blood was collected from AATD (n = 88) or non-AATD COPD patients (n = 10) and healthy controls (HC) (n = 40). Neutrophils were challenged with C3d, and degranulation was assessed by Western blotting, ELISA, or fluorescence resonance energy transfer (FRET) substrate assays. Ex vivo, C3d levels were increased in plasma (p < 0.0001) and on neutrophil plasma membranes (p = 0.038) in AATD compared to HC. C3d binding to CR3 receptors triggered primary (p = 0.01), secondary (p = 0.004), and tertiary (p = 0.018) granule release and increased CXCL8 secretion (p = 0.02). Ex vivo plasma levels of bactericidal-permeability-increasing-protein (p = 0.02), myeloperoxidase (p < 0.0001), and lactoferrin (p < 0.0001) were significantly increased in AATD patients. In endothelial cell scratch wound assays, C3d significantly decreased cell migration (p < 0.0001), an effect potentiated by neutrophil degranulated proteins (p < 0.0001). In summary, AATD patients had increased C3d in plasma and on neutrophil membranes and, together with neutrophil-released granule enzymes, reduced endothelial cell migration and wound healing, with potential implications for AATD-related vasculitis.

scholarly journals ADAMTS-1 and Syndecan-4 intersect in the regulation of cell migration and angiogenesis

2019 ◽  
Author(s):  
Jordi Lambert ◽  
Kate Makin ◽  
Sophia Akbareian ◽  
Robert Johnson ◽  
Stephen D Robinson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Ex Vivo ◽  
Aortic Ring ◽  
Vascular Endothelial ◽  
Collagen Matrices ◽  
Functional Convergence ◽  
Integrin Α5 ◽  
Endothelial Growth Factor

AbstractThe extracellular proteoglycanase ADAMTS-1 has critical roles in organogenesis and angiogenesis. We demonstrate here the functional convergence of ADAMTS-1 and the transmembrane heparan sulfate proteoglycan syndecan-4 in influencing adhesion, migration, and angiogenesis in vitro. Knockdown of ADAMTS-1 in fibroblasts and endothelial cells resulted in a parallel reduction in cell surface syndecan-4 that was not due to altered syndecan-4 expression or internalization, but was attributable to increased expression and activity of matrix metalloproteinase-9 (MMP-9), a known syndecan-4 sheddase. Knockdown of either syndecan-4 or ADAMTS-1 led to enhanced endothelial cell responses to exogenous vascular endothelial growth factor (VEGF), and increased microvessel sprouting in ex vivo aortic ring assays, correlating with reduced ability of the cells to sequester VEGF. On fibronectin but not type 1 collagen matrices, endothelial cells with knockdown of either ADAMTS-1 or syndecan-4 elicited increased migration and showed similarly altered focal adhesion (FA) morphologies, with a higher proportion of larger FA’s and formation of long fibrillar integrin α5-containing FA’s. However, integrin α5-null endothelial cells also displayed enhanced migration in response to ADAMTS-1/syndecan-4 knockdown, indicating that integrin α5 was not the mediator of the altered migratory behaviour. Plating of naïve endothelial cells on cell-conditioned matrix from ADAMTS-1/syndecan-4 knockdown cells demonstrated that the altered behaviour was matrix dependent. Fibulin-1, a known ECM co-factor of ADAMTS-1, was expressed at reduced levels in ADAMTS-1/syndecan-4 knockdown cells. These findings support the notion that ADAMTS-1 and syndecan-4 are functionally interconnected in regulating cell migration and angiogenesis, via the involvement of MMP-9 and fibulin-1 as collaborators

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