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Jennifer A Tran, Xiaoqing Q Guo, Audrey E K Hutcheon, James D Zieske; Effect of TGF-β1 and -β3 on CD63 expression in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1933.
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© ARVO (1962-2015); The Authors (2016-present)
Exosomes are cell-derived microvesicles that have become the subject of increasing interest in recent years since they may play a role in protein, mRNA, and miRNA transport, as well as in wound repair. Studies in our lab show that transforming growth factor-β1 and -β3 (T1 and T3, respectively) are instrumental to corneal wound healing. In this study, we treated human corneal fibroblast (HCF) samples with T1 or T3 to determine their effect on the expression of CD63, a marker of exosomes.
HCF were isolated and cultured in basic medium (BM: EMEM with 10% FBS and Vitamin C) in various conditions for 1 day or 4 weeks: 1) Control: BM only, 2) T1: BM + T1, and 3) T3: BM + T3. Due to the length of time in culture and the medium conditions, the 4-week cultures stratify and accumulate their own extracellular matrix. At the appropriate time, the samples were processed for immunofluorescence (IF) with anti-CD63 and a nuclear counterstain, either DAPI or TOPRO3. Following visualization of both sample sets, the ratio of CD63 expression to nuclei was quantified. Unwounded human corneas were also examined by IF, and negative controls in which the primary antibody was omitted were routinely run with all experiments.
Compared to the 4-week control, the 4-week T1- and T3-treated constructs demonstrated a significant decrease in CD63 expression (p<0.0001), with no significant difference between the latter two conditions. Also, cytoplasmic CD63 expression was apparent in ~1/4 of the cells in the T1- or T3-treated constructs, compared to ~1/2 of the cells in the control. In the 1-day HCFs, however, virtually all of the cells expressed CD63 under all conditions, with no significant difference. Unwounded human corneas expressed low levels of CD63.
As observed with the 1-day samples, isolation and cultivation of HCF appears to be accompanied by a large increase in CD63 (exosomal) expression compared to keratocytes in situ; however, the number of HCF expressing CD63 dramatically decreased upon both T1 and T3 treatment in our 3D constructs. One result from our previous studies indicated that T1 and T3 stimulated the assembly of extracellular matrix. Whether it is the matrix itself or the exposure to T1 or T3 that is having an effect on CD63 expression is currently under investigation.
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