670nm light exposure increases the number of exosomes in the vessel bath and the number of endosomes in endothelial cells
Authors: weihrauch dorothee , Lindemer Brian , Krolikowski John G. , Keszler Agnes , Lohr Nicole 
Published: June 2021

In previous studies we demonstrated endothelial dependent vasodilation at 670nm red/near infrared light (670nm light) in healthy vessels and db/db mice related to S‐nitrosothiol release. Furthermore, the bath solution from intact 670nm light stimulated vessels could dilate 670nm light naïve vessels in a NO dependent manner. Therefore, we propose that 670nm light treatment induces endosome formation in endothelial cells (BAEC) and exosome release.

METHODS

BAEC were cultured on 4 chamber slides and treated with 100mW of 670nm light for 40seconds. Cells were immediately fixed and processed. BAEC were labeled with CD63, caveolin‐1, and FM95‐5 as endosome markers. Dimethyl‐1‐Pyrroline‐N‐Oxide (DMPO) was used to trap free radicals to form a nitroxide radical adduct which decays to produce a stable nitrone, which is recognized by the anti DMPO antibody. This enables us to recognize free radicals.

Exosomes in the vessel bath of murine isolated facialis arteries after 670nm light exposure were counted with a nanosight particle counter and characterized by immunofluorescence. Briefly, the vessel bath was ultra‐centrifuged, the supernatant discarded, and the pellet was either analyzed by nanosight or cytospun onto slides. To identify exosomes, we labeled the cytospin with the styryl peptide FM95‐5 for lipid content, DMPO antibody to identify nitrone radicals in exosomes and von Willebrand Factor (vWF) to identify the origin of the exosomes.

RESULTS

BAEC exhibited higher fluorescence levels of the endosomal marker caveolin‐1 and CD63 after 670nm light treatment (Caveolin‐1: control 95.23 ± 2.73 vs 670nm light 111.53 ± 3.27, CD63: control 21.09 ± 0.74 vs 670nm light 62.47 ± 5.73). Light treatment also significantly increased DMPO (63.67 ± 6.78 vs. 670nm light 127.81 ± 14.87), FM5‐95 (control 8.33 ± 1.18 vs 670nm light 21.76 ± 1.92) and caveolin‐1(control 95.27 ± 2.73 vs 670nm light 111.53 ± 3.27) fluorescence intensity The number of exosomes in the vessel bath was increased after the 670nm light as demonstrated by nanosight (control 3.8 × 107 ± 410,000 vs. 670nm light 6.1×107 ± 294,000). DMPO fluorescence intensity was increased after 670nm light treatment in exosomes (control 87.43 ± 15.24 vs. 670nm light 133.99 ± 11.3) and endosomes (control 63.67 ± 6.78 vs. 670nm light 127.816 ± 14), and about 30% of FM 95‐5 positive exosomes were of endothelial cell origin as indicated by vWF positive labeling.

In CONCLUSION 670nm light treatment induces endosome formation and exosome release. The intracellular vesicles have endosome and lipid droplet characteristics as demonstrated by CD63 and caveolin‐1 labeling. The endosomes and exosomes contain radical complexes as seen by DMPO labeling.

Support or Funding Information

This work was supported by the United States Veteran Health Administration, 1IK2BX002426 (NL).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.