Mouse Conjunctival Fibroblasts

Promotion of Conjunctival Fibroblast-Mediated Collagen Gel Contraction by Mast Cells through Up-Regulation of Matrix Metalloproteinase Release and Activation

Conjunctival fibroblasts manage ECM homeostasis, critically involved in wound healing and fibrosis, primarily regulated by matrix metalloproteinases (MMPs). Mast cells, prevalent in conjunctival tissues, contribute to allergic reactions and scarring. Utilizing a 3D collagen gel culture system, Kiahimoto et al. investigated fibroblast-mast cell interactions' impact on conjunctival fibroblast contractility and ECM remodeling. Assessments include cell viability, collagen contraction, and MMP activity.

Various numbers of mast cells, alone or with conjunctival fibroblasts, were incubated, and MMP levels were tested using gelatin zymography (Fig. 1). Mast cells alone did not produce MMP-2 or MMP-9, but conjunctival fibroblasts did. Mast cells increased pro-MMP-9 and MMP-2 levels in fibroblasts, linked to the number of mast cells. This suggests mast cells aid pro-MMP-9 release by fibroblasts and help convert pro-MMP-2 to active MMP-2. They explored MMP inhibition on dilation by mast cells. Collagen gels with mast cells or fibroblasts were treated with MMP inhibitor batimastat. Batimastat did not affect fibroblast activity alone but reduced contraction in co-cultures with mast cells (Fig. 2). This implies that mast cell enhancement of contraction depends on MMPs.

Fig. 1. Effect of mast cells on MMP expression by conjunctival fibroblasts (Kishimoto T, Ishida W, et al., 2022).

Fig. 2. Effect of batimastat on collagen gel contraction in co-cultures of conjunctival fibroblasts and mast cells (Kishimoto T, Ishida W, et al., 2022).

Increasing the Single siRNA Layer Load in LbL Nanoparticles Prolonged Gene-Silencing Activity in Primary Conjunctival Fibroblasts

The therapeutic use of RNA interference (RNAi) for gene silencing has emerged as a promising treatment for various diseases due to its specificity and low cost. Despite intense research, gene silencing therapies are limited by inefficient delivery systems that degrade or clear siRNA too rapidly. Tan et al. introduced a novel nanocarrier with a hydroxyapatite core and layers of siRNA and cationic peptide to effectively deliver and sustain siRNA targeting SPARC protein. The delivery system was evaluated using FRET studies and in vitro knockdown assays in fibroblasts.

To create siRNA films with high loading and minimal toxicity, hydroxyapatite (HA) was used as the nanoparticle core with alternating layers of ARG, a polycation, and negatively charged siRNA, forming siRNA-LbL films. This method proved effective for cell entry, maintaining viability, and low cytotoxicity. The amount of siRNA in the nanoparticles influences the duration of gene silencing. Three-layered LbL nanoparticles with increased siRNA loads were tested to silence SPARC in mouse conjunctival cells. Nanoparticles with 1.2 pmol SPARC siRNA per µg HA achieved significant mRNA reduction after 7 days (Fig. 3a) and maintained it for 14 days (Fig. 3b). However, the effect lessened over time due to gradual siRNA release. Type I collagen expression remained unchanged (Fig. 3c and d), signifying the need for adjusting dosing and release for sustained silencing.

Fig. 3. Ncreasing the single siRNA layer load in LbL nanoparticles prolonged gene-silencing activity in primary conjunctival fibroblasts (Tan Y F, Lee Y S, et al., 2018).