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Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice.

ABSTRACT: Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGF?2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGF?2 BALB/cJ mouse model of ocular hypertension. BALB/cJ mice that had been intravitreally injected with an adenovirus (Ad5) expressing a bioactive TGF?2226/228 showed a significant increase in IOP after 2 weeks. When 1?M FUD was injected intracamerally into mice 2 weeks post Ad5-TGF?2 injection, FUD significantly reduced IOP after 2 days. Neither mutated FUD (mFUD) nor PBS had any effect on IOP. Four days after FUD was injected, IOP returned to pre-FUD injection levels. In the absence of TGF?2, intracameral injection of FUD had no effect on IOP. Western blotting of mouse anterior segments expressing TGF?2 showed that FUD decreased fibronectin levels 2 days after intracameral injection (p<0.05) but not 7 days compared to eyes injected with PBS. mFUD injection had no significant effect on fibronectin levels at any time point. Immunofluorescence microscopy studies in human TM (HTM) cells showed that treatment with 2ng/ml TGF?2 increased the amount of EDA+ and EDB+ fibronectin incorporated into fibrils and 2?M FUD decreased both EDA+ and EDB+ fibronectin in fibrils. An on-cell western assay validated this and showed that FUD caused a 67% reduction in deoxycholate insoluble fibronectin fibrils in the presence of TGF?2. FUD also caused a 43% reduction in fibronectin fibrillogenesis in the absence of TGF?2 while mFUD had no effect. These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP.

SUBMITTER: Faralli JA

PROVIDER: S-EPMC7444551 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice.

Faralli Jennifer A JA Filla Mark S MS McDowell Colleen M CM Peters Donna M DM

PloS one 20200821 8

Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGFβ2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGFβ2 BALB/cJ mouse model of ocular hy ...[more]

PMID: 32822410

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Project description:We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.

Project description:We reported earlier that the endocochlear potential (EP) differs between C57BL/6J (B6) and BALB/cJ (BALB) mice, being lower in BALBs by about 10 mV (Ohlemiller et al. Hear Res 220: 10-26, 2006). This difference corresponds to strain differences with respect to the density of marginal cells in cochlear stria vascularis. After about 1 year of age, BALB mice also tend toward EP reduction that correlates with further marginal cell loss. We therefore suggested that early sub-clinical features of the BALB stria vascularis may predispose these mice to a condition modeling Schuknecht's strial presbycusis. We further reported (Ohlemiller et al. J Assoc Res Otolaryngol 12: 45-58, 2011) that the acute effects of a 2-h 110 dB SPL noise exposure differ between B6 and BALB mice, such that the EP remains unchanged in B6 mice, but is reduced by 40-50 mV in BALBs. In about 25 % of BALBs, the EP does not completely recover, so that permanent EP reduction may contribute to noise-induced permanent threshold shifts in BALBs. To identify genes and alleles that may promote natural EP variation as well as noise-related EP reduction in BALB mice, we have mapped related quantitative trait loci (QTLs) using 12 recombinant inbred (RI) strains formed from B6 and BALB (CxB1-CxB12). EP and strial marginal cell density were measured in B6 mice, BALB mice, their F1 hybrids, and RI mice without noise exposure, and 1-3 h after broadband noise (4-45 kHz, 110 dB SPL, 2 h). For unexposed mice, the strain distribution patterns for EP and marginal cell density were used to generate preliminary QTL maps for both EP and marginal cell density. Six QTL regions were at least statistically suggestive, including a significant QTL for marginal cell density on chromosome 12 that overlapped a weak QTL for EP variation. This region, termed Maced (Marginal cell density QTL) supports the notion of marginal cell density as a genetically influenced contributor to natural EP variation. Candidate genes for Maced notably include Foxg1, Foxa1, Akap6, Nkx2-1, and Pax9. Noise exposure produced significant EP reductions in two RI strains as well as significant EP increases in two RI strains. QTL mapping of the EP in noise-exposed RI mice yielded four suggestive regions. Two of these overlapped with QTL regions we previously identified for noise-related EP reduction in CBA/J mice (Ohlemiller et al. Hear Res 260: 47-53, 2010) on chromosomes 5 and 18 (Nirep). The present map may narrow the Nirep interval to a ~10-Mb region of proximal Chr. 18 that includes Zeb1, Arhgap12, Mpp7, and Gjd4. This study marks the first exploration of natural gene variants that modulate the EP. Their orthologs may underlie some human hearing loss that originates in the lateral wall.

Dataset Information

Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice.

Publications

Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice.

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