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Garima Sharma, Maryse Bailly, Steve Brocchini, Peng Tee Khaw; Macrophages Promote Conjunctival Fibroblast Contraction And Nullify The Effect Of Anti-Scarring Agents. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5618.
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© ARVO (1962-2015); The Authors (2016-present)
We have developed a novel in vitro model that incorporates two important aspects of fibrosis – inflammation and fibroblast-mediated tissue contraction. This study examined the effect of inflammatory stimulus provided by macrophages on the efficiency of anti-scarring drugs on fibroblast-populated collagen gel contraction.
Macrophages derived from U937 monocyte-like human cell line were co-cultured with human Tenon’s capsule fibroblasts in three-dimensional collagen gels. The gels were treated with a broad spectrum MMP inhibitor (GM6001) or Rac1 inhibitor (NSC23766). Additionally, fibroblasts were treated with different doses of Mitomycin-C (MMC) for 5 minutes, and then seeded in collagen gels at different fibroblast:macrophages ratios (1:2 or 1:5). Contraction was calculated from digital photographs using ImageJ.
Broad-spectrum MMP inhibitor prevents contraction in fibroblast populated collagen gels. However, in co-culture conditions (ratio 1:2) macrophages were able to completely overcome the effect of the inhibitor (Fig. 1A).Similarly, treatment with Rac1 inhibitor for the first 24-hours also prevented fibroblast-mediated contraction for up to 4 days. The Rac1 inhibitor also successfully blocked the ability of macrophages to stimulate fibroblast-mediated gel contraction when the fibroblast:macrophage ratio was low (1:2). However, increasing the ratio to 1:5 nullified the effect of the inhibitor (Fig. 1B).When fibroblasts were seeded alone, MMC (0.2 mg/ml) successfully inhibited contraction. With a fibroblast:macrophage ratio of 1:2, MMC at lower doses did not prevent contraction and a higher dose of MMC (1 mg/ml) was required to completely inhibit contraction. Furthermore, a higher ratio of macrophages (1:5) completely prevented the effect of MMC on contraction, even at the highest dose (Fig. 2).
The fibroblast sensitivity to anti-scarring treatment is significantly altered in presence of inflammation. This co-culture model for tissue contraction and inflammation in fibrosis can be used to study the effect of inflammatory stimulus on the efficacy of anti-scarring drugs. This has implications for the use and dosing of anti-scarring drugs in situations where different degrees of inflammation are present.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Macrophages decrease sensitivity to anti-scarring drugs (A) MMP inhibitor (B) Rac1 inhibitor (*p<0.05)
Macrophages can overcome the effect of high doses of MMC
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