226 EFFECTS OF MICROVESICLES SECRETED FROM EQUINE AMNIOTIC-DERIVED PROGENITOR CELLS ON IN VITRO LIPOPOLYSACCHARIDE-TREATED TENDON AND ENDOMETRIAL CELLS

Abstract

Administration of horse amniotic mesenchymal cell conditioned medium (AMC-CM) improves the in vivo recovery of spontaneous equine tendon lesions. This effect may involve paracrine mechanisms whose nature remains unknown. It has recently been demonstrated that microvesicles (MV) released from cells are an integral component of cell-to-cell communication during tissue regeneration. Aims of this study were to investigate the presence and type of MV secreted by AMC using Nanosight instrument (Malvern Instruments, Malvern, UK) and transmission electron microscopy (TEM) and the incorporation of MV in equine tendon and endometrial cells by fluorescence semiquantitative analysis. Tendon cells were used to understand the in vitro role of MV on stressed cells compared with the in vivo results previously obtained, while the endometrial cells were investigated in view of the prospective use of AMC-CM or MV in in vivo inflammatory endometrial diseases. Moreover, the ability of MV to counteract in vitro inflammation of tendon and endometrial cells induced by lipopolysaccharide (LPS) was also evaluated. The MV were obtained by ultracentrifugation at 100 000 × g for 1 h at 4°C of the media obtained by culturing AMC isolated from 3 different placentas. Tendon and endometrial cells were obtained from collagenase digestion for 17 and 3 h, respectively and cultured in HG-DMEM with 10% fetal calf serum. To study the ability of tendon and endometrial cells to incorporate MV, a dose-response curve was performed adding 10, 20, 30, 40, and 50 × 10⁶ MV mL^–1 labelled with PKH-26 for 24, 48, and 72 h. The uptake of MV was evaluated by an Olympus BX51 microscope (Olympus, Tokyo, Japan) equipped with software for image acquisition. A dose-response curve of LPS was also investigated by apoptotic and MTT tests showing that 100 ng mL^–1 at 48 h on tendon cells and 10 ng at 24 h on endometrial cells were the doses and times most effective in inducing cellular stress. RT-qPCR expression of pro-inflammatory genes such as metallopeptidase (MMP) 1 and 13 was evaluated in the in vitro LPS stress by Mann-Whitney U-test. Results by Nanosight Instrument showed that AMC secrete MV in the range of 100 to 200 nm; TEM showed budding of the AMC membrane, proving that these MV fall within the shedding vesicles category. The same semiquantitative fluorescence uptake signal was obtained when 50 × 10⁶ MV were incorporated at 24 h, or 40 × 10⁶ MV at 48 h, and 30 × 10⁶ MV at 72 h, suggesting that an inverse correlation between concentration and time was found in MV uptake equally by tendon and endometrial cells. The MV induced a significant (P < 0.05) down-regulation of MMP1 and MMP13 expression in both cell lines after in vitro LPS stress. Our data suggest that these MV can be incorporated in tendon and endometrial cells and have a role in modulating inflammatory genes in vitro.