Amphibian thrombocyte‐derived extracellular vesicles, including microRNAs, induce angiogenesis‐related genes in endothelial cells

Abstract

Thrombocytes circulate in the blood of nonmammalian vertebrates and are involved in hemostasis; however, many detailed characteristics of thrombocytes remain unclear. Recently, we established an amphibian thrombocyte cell line. Here, we report the finding that thrombocytes produce integrin alpha IIb (CD41)‐positive extracellular vesicles (EVs), which include microRNAs (miRs). Flow cytometric analysis showed the expression of CD41+ and phosphatidylserine on the surface of EVs. Nanotracking analysis showed that these CD41+ EVs were approximately 100 nm in diameter. As CD41+ EVs were also observed from African clawed frogs, the production of CD41+ EVs might be common to amphibians. Microarray analysis showed that the CD41+ EVs contain many kinds of miRs. These CD41+ EVs were phagocytosed by endothelial cells and macrophages. qPCR analysis showed that many angiogenesis‐related genes were up‐regulated in CD41+ EV‐treated endothelial cells. Over‐expression of some miRs in the CD41+ EVs increased the proliferation of endothelial cells. These results indicated that thrombocytes produced CD41+ EVs, including miRs, that were received by endothelial cells to induce the expression of angiogenesis‐related genes. These results indicated that the CD41+ EVs produced from thrombocytes act as signaling molecules to repair damaged blood vessels.

CD41+ extracellular vesicles produced by avian thrombocytes contain microRNAs

“…For FCM analysis, purified EVs were adsorbed to aldehyde/sulfate latex beads and then reacted with FITC‐conjugated αCD41 or anti‐CD9 antibody (αCD9). This method enabled the analysis of small EVs compared to the liquid drops needed for FCM (Sugimoto & Toume, 2021). The specific FITC‐conjugated αCD41 antibody could detect EVs adsorbed to latex beads, while an Alexa488‐conjugated IgG isotype control did not react with EV‐adsorbed beads (Figure 2e).…”

“…In this study, we compared avian miR data with amphibian miR data. The amphibian miR data were derived from a past report (Sugimoto & Toume, 2021). The data indicated that 129 miRs were identical between amphibians and avians (Figure 4a).…”

“…The expression value of each gene relative to that of β‐actin was calculated. The details are described in our previous report (Sugimoto & Toume, 2021). The primers are summarized in Table S2.…”

“…Blood cells, spleen cells, and bone marrow cells were stained with FITC‐conjugated αCD41 and then analyzed by FCM as described previously (Sugimoto & Toume, 2021). For further preparation of CD41‐positive cells, spleen cells were separated by discontinuous density gradient centrifugation using Percoll (Cytiva, Tokyo, Japan).…”

“…Recently, we reported that amphibian CD41 + thrombocytes produce CD41 + EVs containing miRs. The CD41 + EVs were taken up by macrophages and endothelial cells and changed the gene expression in the target cells via their miR cargo (Sugimoto & Toume, 2021). These observations suggested that the thrombocytes of other animal classes may also produce CD41 + EVs.…”