Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Steinritz, Dirk; Schmidt, Annette; Balszuweit, Frank; Thiermann, Horst; Ibrahim, Marwa A.; Bölck, Birgit; Bloch, Wilhelm

doi:10.3791/52768

Cited by 7 publications

(6 citation statements)

References 16 publications

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“…Potential correlation between endothelial Myct1 expression and the immune output in the human tumor microenvironment was evaluated by analyzing cancer datasets using the CIBERSORT deconvolution algorithm (45). Using various in vitro assays, the role(s) of Myct1 in ECs and the underlying mechanisms for regulating antitumor immunity were rigorously investigated (17,(77)(78)(79). Last, using both the KO mice and siRNA-peptide nanoparticle-mediated approach (17), the efficacy of Myct1 inhibition for treating tumors in combination with anti-PD1 immunotherapy was evaluated.…”

Section: Methodsmentioning

confidence: 99%

Dual role of endothelial Myct1 in tumor angiogenesis and tumor immunity

et al. 2021

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The cross-talk between angiogenesis and immunity within the tumor microenvironment (TME) is critical for tumor prognosis. While pro-angiogenic and immunosuppressive TME promote tumor growth, anti-angiogenic and immune stimulatory TME inhibit tumor progression. Therefore, there is a great interest in achieving vascular normalization to improve drug delivery and enhance antitumor immunity. However, anti–vascular endothelial growth factor (VEGF) mechanisms to normalize tumor vessels have offered limited therapeutic efficacies for patients with cancer. Here, we report that Myct1, a direct target of ETV2, was nearly exclusively expressed in endothelial cells. In preclinical mouse tumor models, Myct1 deficiency reduced angiogenesis, enhanced high endothelial venule formation, and promoted antitumor immunity, leading to restricted tumor progression. Analysis of The Cancer Genome Atlas (TCGA) datasets revealed a significant (P < 0.05) correlation between MYCT1 expression, angiogenesis, and antitumor immunity in human cancers, as suggested by decreased FOXP3 expression and increased antitumor macrophages in patients with low MYCT1 expression. Mechanistically, MYCT1 interacted with tight junction protein Zona Occludens 1 and regulated Rho GTPase-mediated actin cytoskeleton dynamics, thereby promoting endothelial motility in the angiogenic environment. Myct1-deficient endothelial cells facilitated trans-endothelial migration of cytotoxic T lymphocytes and polarization of M1 macrophages. Myct1 targeting combined with anti-PD1 treatment significantly (P < 0.05) increased complete tumor regression and long-term survival in anti-PD1–responsive and –refractory tumor models in mice. Our data collectively support a critical role for Myct1 in controlling tumor angiogenesis and reprogramming tumor immunity. Myct1-targeted vascular control, in combination with immunotherapy, may become an exciting therapeutic strategy.

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Section: Methodsmentioning

confidence: 99%

Dual role of endothelial Myct1 in tumor angiogenesis and tumor immunity

et al. 2021

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“…Mitochondrial membrane potential was assessed by using tetramethylrhodamine methyl ester perchlorate (TMRM, T5428) fluorescent probe. TMRM is a cationic lipophilic, nontoxic and highly fluorescent compound which has been used to analyse the ΔΨm in somatic cells (Buckman & Reynolds, ; Kvetny, Wilms, Pedersen, & Larsen, ; Labios et al, ; Steinritz et al, ; Ward, Rego, Frenguelli, & Nicholls, ), bovine sperm (Aguila, Arias, Vargas, Zambrano, & Felmer, ) and human sperm (Uribe et al, ). Briefly, 1 μl of TMRM (250 µM stock solution) and 2 μl of SYTOX green (20 µM stock solution, Molecular Probes, Eugene, OR, USA) were added to 500 μl of sperm suspension (2 × 10 6 sperm/ml) and incubated for 30 min at 38.5°C in darkness ( n = 3 replicates for each stallion).…”

Section: Methodsmentioning

confidence: 99%

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Arroyo-Salvo

Sanhueza

Fuentes

et al. 2018

Reprod Domestic Animals

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Conventional in vitro fertilization has not yet been implemented in the equine species. One of the main reasons has been the inability to develop a culture medium and incubation conditions supporting high levels of stallion sperm capacitation and hyperactivation in vitro. Although different culture media have been used for this purpose, human tubal fluid (HTF) medium, widely used in the manipulation of human and mice gametes, has not been reported so far in stallion sperm culture. The first part of this study aimed to compare HTF and Whitten's media on different stallion sperm quality and capacitation variables. Additionally, the effect of procaine, aminopyridine and caffeine in both media was evaluated on sperm motility parameters at different incubation times. Integrity and destabilization of the plasma membrane were evaluated by merocyanine 540/SYTOX Green (MC540), mitochondrial membrane potential (∆Ψm) using tetramethylrhodamine methyl ester perchlorate (TMRM), acrosome membrane integrity by PNA/FITC and tyrosine phosphorylation by P-tyrosine mouse mAb conjugated to Alexa Fluor® by flow cytometry. Motility parameters were evaluated using the integrated semen analysis system (ISAS®). We found no differences between Whitten's and HTF media and incubation time in terms of sperm viability, uninduced acrosome membrane damage or mitochondrial membrane potential at 30- and 120-min incubation. Membrane fluidity (MC540) increased in both media at 30- and 120-min incubation compared to noncapacitating conditions. Similarly, tyrosine phosphorylation increased in both media in capacitating conditions at 2- and 4-hr incubation compared to noncapacitating conditions. Although procaine showed the best result in terms of sperm hyperactivated motility in both media, aminopyridine also showed parameters consistent with the hyperactivation including an increase in curvilinear velocity and decrease in straightness. In conclusion, HTF medium and aminopyridine equally support capacitation-related parameters in stallion sperm.

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“…Tetramethylrhodamine methyl ester perchlorate (TMRM) is a cationic lipophilic, nontoxic and highly fluorescent compound widely used to analyse ΔΨm in somatic cells (Buckman & Reynolds, ; Kvetny, Wilms, Pedersen, & Larsen, ; Labios et al., ; Steinritz et al., ; Ward, Rego, Frenguelli, & Nicholls, ) and in bovine spermatozoa (Mizrahi & Breitbart, ), but to our knowledge it has not been used in human spermatozoa. In this context, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for ΔΨm measurement in human spermatozoa.…”

Section: Introductionmentioning

confidence: 99%

Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa

et al. 2017

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Mitochondrial membrane potential (ΔΨm) is an indicator of sperm quality and its evaluation complements the standard semen analysis. The fluorescent dye JC-1 has been widely used to assess sperm ΔΨm; however, some problems have been detected under certain experimental conditions. Another fluorescent compound, tetramethylrhodamine methyl ester perchlorate (TMRM), has been used in somatic cells and bovine spermatozoa but not in human spermatozoa. TMRM accumulates in hyperpolarised mitochondria and the fluorescence intensity of this compound correlates with ΔΨm. Thus, the aim of this study was to evaluate and validate the usefulness of the fluorescent dye TMRM for measuring sperm ΔΨm. The results showed that TMRM accurately detects sperm populations displaying either high or low ΔΨm. Moreover, TMRM was able to measure sperm ΔΨm under the experimental conditions in which JC-1 had previously presented difficulties. Differences in ΔΨm according to sperm and semen quality were properly detected and a positive correlation between ΔΨm and conventional semen parameters was observed. Finally, a positive correlation was found between the ΔΨm measurement by TMRM and by the widely used JC-1. In conclusion, TMRM is a simple, time-effective method, easy to set in laboratories equipped with flow cytometry technology, and can accurately detect changes in ΔΨm with efficiency comparable to JC-1 without its limitations.

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Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Cited by 7 publications

References 16 publications

Dual role of endothelial Myct1 in tumor angiogenesis and tumor immunity

Dual role of endothelial Myct1 in tumor angiogenesis and tumor immunity

Effect of human tubal fluid medium and hyperactivation inducers on stallion sperm capacitation and hyperactivation

Use of the fluorescent dye tetramethylrhodamine methyl ester perchlorate for mitochondrial membrane potential assessment in human spermatozoa

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