Mechanism of fibrosis inhibition in laser induced choroidal neovascularization by doxycycline

To explore the mechanisms underlying doxycycline suppression of fibrosis in laser-induced choroidal neovascularization (LCNV), C57BL/6J male mice (aged from 6 to 8 weeks) received intraperitoneal injections of PBS/doxycycline solution from one day before laser injury until they were sacrificed. Leakage was assessed by FA, and CNV (stained by IB4) or fibrosis (stained by collagen type I) size was measured. The percentage of Pan-keratin+α-SMA+ cells was counted in the eyes' cryostat sections by immunohistochemistry. qPCR was used to measure the mRNA of markers of pan-macrophages, M1 and M2-type macrophages (M1 and M2), markers of EMT, and markers in the downstream of STAT6 signaling. Western blotting was used to analyze the expression of Arg-1, α-SMA, E-cadherin, pSTAT6 and STAT6. Our data showed that doxycycline inhibited leakage from CNV, areas of CNV on day 7 and day 14, and suppressed fibrosis, and the ratio of fibrotic/angiogenic areas during day 7 to day 35. We also showed attenuation of EMT in the doxycycline group. The percentage of Pan-keratin+α-SMA+ cells was lower in the doxycycline group than in the control group. The mRNA and protein levels of mesenchymal markers were downregulated in the doxycycline group, while the epithelial marker was upregulated. In addition, our data showed that the protein expression of Arg-1, the mRNA expression of M1 and M2-markers, were both inhibited by doxycycline, while the level of pan-macrophages (f4/80) showed no significant difference in two groups. Finally, our results showed that doxycycline was able to modulate the STAT6 signaling in transcript and protein levels. Accordingly, we suggested that the mechanism of doxycycline-mediated inhibition of fibrosis in CNV occurs through the STAT6 pathway. •Doxycycline inhibits CNV and following fibrosis.•Doxycycline inhibits epthelial-mesenchymal transition.•Doxycycline inhibits macrophage polarization toward M2 during CNV and subsequent fibrosis.•STAT6 signaling may play important role in fibrosis inhibition of doxycycline.