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. 2023 Jul-Aug;37(4):1680-1687.
doi: 10.21873/invivo.13254.

Reduced Production of JAK2V617F-positive Microparticles at Mild Hypothermia

Hilal Hekimoglu  1   2 , Esra Nur Demirtas  1   2 , Selcuk Sozer  3
Affiliations

Reduced Production of JAK2V617F-positive Microparticles at Mild Hypothermia

Hilal Hekimoglu et al. In Vivo. 2023 Jul-Aug.

Abstract

Background/aim: The Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise from abnormal growth of blood cells in the bone marrow. Patients with MPNs are at increased risk for life-threatening thromboembolic complications. The detection of JAK2V617F in endothelial cells (ECs) brought a new perspective to the research of thromboembolic events. However, the mechanisms by which the mutation contributes to risk have yet to be entirely understood. Consequently, the objective of this study was to investigate how JAK2V617F impacts endothelial cells by considering thermoregulation.

Materials and methods: We applied our previously created model for EC that was genetically modified with JAK2 wild type (WT)-GFP and JAK2V617F-GFP lentiviruses; the cells were cultured for 48 h at 37°C for normothermia and 32°C for mild hypothermia. We examined the effect of thermoregulation on infection efficiency and the expression of cell surface markers, including endothelial protein C receptor (EPCR), thrombomodulin (TM), and tissue factor (TF), which are related to the coagulation pathways. Furthermore, the microparticle production from the genetically modified EC (EMPs) was analyzed.

Results: We found suppression of the expression of coagulation factors, including EPCR, TM, and TF in JAK2V617F positive ECs under mild hypothermia. JAK2V617F-positive ECs showed slightly higher EMP production under mild hypothermia.

Conclusion: Although the molecular mechanisms of the thermal effects on the tumor microenvironment with JAK2V617F and its effect on EMP production and coagulation are not known yet, the therapy-oriented effect of thermoregulation might be considered in future studies.

Keywords: JAK2V617F; endothelial cells; extracellular vesicles; hypothermia; microparticles; thrombosis.

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Conflict of interest statement

The Authors have no conflicts of interest to declare in relation to this study.

Figures

Figure 1
Figure 1. Thermoregulation model for microparticle analysis. A) Schematic of the experimental design and the lentiviral vectors used in the study. B) Lentiviral infection efficiency decreases under mild hypothermic conditions. The infection efficiency of JAK2WT and JAK2V617F lentiviruses was analyzed under normothermia and mild hypothermia. Error bars show standard deviation. ***p<0.001; **p<0.01.
Figure 2
Figure 2. Cell surface expression analysis of genetically modified HUVECs under normothermia and mild hypothermia. A) The cell surface expression of EPCR on genetically modified HUVECs under normothermia and mild hypothermia: EPCR expression in JAK2WTand JAK2V617F-HUVECs under normothermia and mild hypothermia. Error bars show standard deviation. **p<0.01. B) The cell surface expression of TM on genetically modified HUVECs under normothermia and mild hypothermia conditions: TM expression in JAK2WT- and JAK2V617F-HUVECs under normothermia and mild hypothermia. Error bars show standard deviation. *p<0.05, **p<0.01. C) The Cell Surface expression of TF on genetically modified HUVECs under normothermia and mild hypothermia conditions: TF expression in JAK2WT- and JAK2V617F-HUVECs under normothermia and mild hypothermia. Error bars show standard deviation. *p<0.05.
Figure 3
Figure 3. The microparticle surface expression analysis under normothermia and mild hypothermia. A) The expression of PECAM-1 on EMPs under normothermia and mild hypothermia conditions. The expression of PECAM-1 in JAK2WT- and JAK2V617F-positive endothelial microparticles (EMP) collected from JAK2WT- and JAK2V617F- HUVECs. The analysis was performed under normothermic and mild hypothermic conditions. Error bars show standard deviation. ****p<0.0001, *p<0.05. B) The expression of TF on EMPs under normothermia and mild hypothermia conditions. Cytosolic TF e levels in JAK2WT- and JAK2V617F- positive endothelial microparticles (EMP) collected from JAK2WT- and JAK2V617F-HUVECs. The analysis was performed under normothermic and mild hypothermic conditions. Error bars show standard deviation. ***p<0.0001, *p<0.05.
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