一氧化氮合酶抑制的内皮细胞衍生的微泡促进细胞功能障碍。

Microvesicles Derived from Nitric Oxide Synthase-Inhibited Endothelial Cells Promote Cell Dysfunction.

作者信息

Garcia Vinicius P, Stockelman Kelly A, Levy Ma'ayan V, Fandl Hannah K, Goulding Anabel, Hijmans Jamie G, Ruzzene Samuel T, Berry Auburn R, Greiner Jared J, DeSouza Christopher A

机构信息

Integrative Vascular Biology Laboratory, Department of Integrative Physiology, University of Colorado Boulder, Boulder, Colorado, USA.

出版信息

J Vasc Res. 2025;62(1):10-21. doi: 10.1159/000542280. Epub 2024 Dec 10.

DOI:10.1159/000542280

PMID:39657607

Abstract

INTRODUCTION

The aims of this study were to determine (1) whether endothelial nitric oxide synthase (eNOS) inhibition stimulates endothelial microvesicles (EMVs) release and (2) the effect of EMVs derived from eNOS-inhibited cells on endothelial cell eNOS, inflammation, apoptosis, and tissue-type plasminogen activator (t-PA).

METHODS

Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor (NG-nitro-l-arginine methyl ester [L-NAME], 300 µM) for 24 h. EMVs from untreated and L-NAME-treated cells were isolated, quantified, and exposed to HUVECs for 24 h.

RESULTS

eNOS-inhibited cells released significantly higher EMVs than untreated cells (81 ± 13 vs. 41 ± 15 EMV/μL; p = 0.005). Expression of total eNOS (97.1 ± 16.4 vs. 157.5 ± 31.2 arbitrary units [AUs]; p = 0.01), p-eNOS (4.9 ± 1.2 vs. 9.1 ± 12.6 AUs; p = 0.02), and NO production (5.0 ± 0.8 vs. 7.0 ± 1.3 µmol/L; p = 0.04) were significantly lower in cells treated with EMVs from L-NAME-treated cells. L-NAME-derived EMVs induced significantly higher IL-6 (38.3 ± 10.3 vs. 21.0 ± 3.8 pg/mL; p = 0.01) and IL-8 (38.9 ± 7.0 vs. 27.2 ± 6.2 pg/mL; p = 0.04) production concurrent with higher expression of p-NF-κB p65 (Ser536) (9.7 ± 1.6 vs. 6.1 ± 1.2 AUs; p = 0.01). Expression of activated caspase-3 was higher (9.5 ± 1.1 vs. 6.4 ± 0.4 AUs) and t-PA lower (24.2 ± 4.3 vs. 36.2 ± 8.4 AUs; p = 0.04) in cells treated with L-NAME-derived EMVs.

CONCLUSION

eNOS inhibition induces an increase in EMV release and an EMV phenotype with adverse cellular effects.

摘要

引言

本研究的目的是确定（1）内皮型一氧化氮合酶（eNOS）抑制是否会刺激内皮微泡（EMV）释放，以及（2）源自eNOS抑制细胞的EMV对内皮细胞eNOS、炎症、凋亡和组织型纤溶酶原激活剂（t-PA）的影响。

方法

用人脐静脉内皮细胞（HUVECs）与eNOS抑制剂（NG-硝基-L-精氨酸甲酯[L-NAME]，300μM）处理24小时。从未处理和L-NAME处理的细胞中分离、定量EMV，并将其暴露于HUVECs 24小时。

结果

与未处理的细胞相比，eNOS抑制的细胞释放的EMV显著更高（81±13对41±15个EMV/μL；p = 0.005）。用源自L-NAME处理细胞的EMV处理的细胞中，总eNOS的表达（97.1±16.4对157.5±31.2任意单位[AUs]；p = 0.01）、p-eNOS（4.9±1.2对9.1±12.6 AUs；p = 0.02）和NO生成（5.0±0.8对7.0±1.3μmol/L；p = 0.04）显著降低。源自L-NAME的EMV诱导显著更高的IL-6（38.3±10.3对21.0±3.8 pg/mL；p = 0.01）和IL-8（38.9±7.0对27.2±6.2 pg/mL；p = 0.04）生成，同时p-NF-κB p65（Ser536）的表达更高（9.7±1.6对6.1±1.2 AUs；p = 0.01）。在用源自L-NAME的EMV处理的细胞中，活化的半胱天冬酶-3的表达更高（9.5±1.1对6.4±0.4 AUs），而t-PA更低（24.2±4.3对36.2±8.4 AUs；p = 0.04）。