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Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature
Kasra Khalaj, … , Madhuri Koti, Chandrakant Tayade
Kasra Khalaj, … , Madhuri Koti, Chandrakant Tayade
Published September 19, 2019
Citation Information: JCI Insight. 2019;4(18):e128846. https://doi.org/10.1172/jci.insight.128846.
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Research Article Reproductive biology

Extracellular vesicles from endometriosis patients are characterized by a unique miRNA-lncRNA signature

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Abstract

With multifactorial etiologies, combined with disease heterogeneity and a lack of suitable diagnostic markers and therapy, endometriosis remains a major reproductive health challenge. Extracellular vesicles (EVs) have emerged as major contributors of disease progression in several conditions, including a variety of cancers; however, their role in endometriosis pathophysiology has remained elusive. Using next-generation sequencing of EVs obtained from endometriosis patient tissues and plasma samples compared with controls, we have documented that patient EVs carry unique signatures of miRNAs and long noncoding RNAs (lncRNAs) reflecting their contribution to disease pathophysiology. Mass spectrophotometry–based proteomic analysis of EVs from patient plasma and peritoneal fluid further revealed enrichment of specific pathways, as well as altered immune and metabolic processes. Functional studies in endometriotic epithelial and endothelial cell lines using EVs from patient plasma and controls clearly indicate autocrine uptake and paracrine cell proliferative roles, suggestive of their involvement in endometriosis. Multiplex cytokine analysis of cell supernatants in response to patient and control plasma–derived EVs indicate robust signatures of important inflammatory and angiogenic cytokines known to be involved in disease progression. Collectively, these findings suggest that endometriosis-associated EVs carry unique cargo and contribute to disease pathophysiology by influencing inflammation, angiogenesis, and proliferation within the endometriotic lesion microenvironment.

Authors

Kasra Khalaj, Jessica E. Miller, Harshavardhan Lingegowda, Asgerally T. Fazleabas, Steven L. Young, Bruce A. Lessey, Madhuri Koti, Chandrakant Tayade

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Figure 5

Uptake of endometriosis patient plasma and peritoneal fluid EVs, as well as endometriotic and endometrial epithelial–derived EV uptake in human endothelial cells and extracellular vesicle, including exosomes, coculture with HUVECs results in increased angiogenesis.

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Uptake of endometriosis patient plasma and peritoneal fluid EVs, as well...
(A–D) Confocal microscopy images of labeled EVs from 12Z (A), EECC (B), HUVEC (C) and negative (raw HUVEC supernatant labeled; no EV purification) (D) at 6 hours in HUVECs. 12Z-labeled EVs were found in moderate to high quantities in the cytoplasm of endothelial cells (A) at 6 hours. EECC-labeled exosomes were detected in high to very high levels in the cytoplasm and perinuclear space in endothelial cells at 6 hours (B). HUVEC-labeled exosomes were detected in low quantities in the cytoplasm and nucleus of endothelial cells at 6 hours (C), and no exosomes were detected in endothelial cells from negative group (D). (E and F) 12Z- and EECC-labeled exosomes accumulated in high quantities in the cytoplasmic, perinuclear, and basal locations of endothelial cells at 12 hours. (G) Confluencies were calculated using the cell proliferation algorithm (Incucyte software) and plotted over 24 hours for each group, with 12Z EVs displaying increased cell proliferation at 10 hours (2-way RM ANOVA). (H) Bright-field images of endometriosis patient plasma EVs cocultured with HUVECs results in increased angiogenesis compared with healthy, fertile control plasma EVs (t test). Branch length and total lengths were plotted and compared between endometriosis patient plasma EVs cocultured with HUVECs compared with healthy, fertile control plasma EVs. Expression data illustrated as mean ± SEM. *P < 0.05. Green dye indicate cytoplasm, blue dye indicate nucleus (DAPI), and red dye indicate fluorescently labeled exosomes. Images representative of n = 3 independent experiments. Scale bar: 15 μm.

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