Regional Differences and Physiologic Behaviors in Peripapillary Scleral Fibroblasts

Abstract

The purpose of this study was to describe the cellular architecture of normal human peripapillary sclera (PPS) and evaluate surface topography's role in fibroblast behavior. METHODS.PPS cryosections from nonglaucomatous eyes were labelled for nuclei, fibrillar actin (FA), and alpha smooth muscle actin (αSMA) and imaged. Collagen fibrils were imaged using second harmonic generation. Nuclear density and aspect ratio of the internal PPS (iPPS), outer PPS (oPPS), and peripheral sclera were determined. FA and αSMA fibril alignment with collagen extracellular matrix (ECM) was determined. PPS fibroblasts were cultured on smooth or patterned membranes under mechanical strain and in the presence of TGFβ1 and 2. RESULTS.The iPPS (7.1 ± 2.0 × 10 −4 , P < 0.0001) and oPPS (5.3 ± 1.4 × 10 −4 , P = 0.0013) had greater nuclei density (nuclei/μm 2 ) than peripheral sclera (2.5 ± 0.8 × 10 −4 ). The iPPS (2.0 ± 0.3, P = 0.002) but not oPPS (2.4 ± 0.4, P = 0.45) nuclei had smaller aspect ratios than peripheral (2.7 ± 0.5) nuclei. FA was present throughout the scleral stroma and was more aligned with oPPS collagen (9.6 ± 1.9 degrees) than in the peripheral sclera (15.9 ± 3.9 degrees, P =0.002). The αSMA fibers in the peripheral sclera were less aligned with collagen fibrils (26.4 ± 4.8 degrees) than were FA (15.9 ± 3.9 degrees, P = 0.0002). PPS fibroblasts cultured on smooth membranes shifted to an orientation perpendicular to the direction of cyclic uniaxial strain (1 Hz, 5% strain, 42.2 ± 7.1 degrees versus 62.0 ± 8.5 degrees, P < 0.0001), whereas aligned fibroblasts on patterned membranes were resistant to strain-induced reorientation (5.9 ± 1.4 degrees versus 10 ± 3.3 degrees, P = 0.21). Resistance to re-orientation was reduced by TGFβ treatment (10 ± 3.3 degrees without TGFβ1 compared to 23.1 ± 4.5 degrees with TGFβ1, P < 0.0001). CONCLUSIONS.Regions of the posterior sclera differ in cellular density and nuclear morphology. Topography alters the cellular response to mechanical strain.

TGFβ treatment alters the scleral fibroblast migratory response to cyclic strain in an ERK-dependent manner

“…Fibroblasts cultured on a surface with grooved topography align with the long axis of the grooves. We previously demonstrated that the combination of TGFβ treatment and cyclic uniaxial strain applied parallel to the direction of these grooves reduced fibroblast cell body alignment with surrounding topography 7 . Here, we expand on those findings by examining fibroblast-ECM alignment at baseline, following 18 hours of cyclic strain (1 Hz, 5% strain), and after a 24-hour recovery period during which the strain was stopped.…”

“…The sclera is relatively paucicellular but contains populations of macrophages, stem cells, and fibroblasts [4][5][6] . Resident fibroblasts are the main scleral cellular component and can be found throughout the sclera with high cytoskeletal alignment to surrounding extracellular matrix (ECM) 7 . Oglesby and colleagues showed fibroblast proliferation after IOP elevation and increased aSMA expression which is a marker for myofibroblasts -a cell type involved in tissue remodeling throughout the body 8 .…”

“…In normal human sclera, we measured a high degree of cellular cytoskeletal alignment with surrounding collagen ECM fibrils; however, alignment was reduced in aSMA-expressing myofibroblasts 7 . When examined in vitro , myofibroblasts oriented with contact guidance cues in the absence of strain.…”

Remodeling of the Lamina Cribrosa: Mechanisms and Potential Therapeutic Approaches for Glaucoma

“…Interestingly, this pattern correlates with distribution and alignment of scleral fibroblasts. Fibroblast density increases in proximity to the ONH, and fibroblast projections are highly aligned with collagenous structures [ 112 ]. There is also limited evidence to suggest that such fibroblast density gradients may exist in mouse as well [ 113 ].…”

“…Given this precise alignment of fibroblasts and collagen, these cells likely play a role in the detection of tissue stretch. The reaction of fibroblasts may also differ based on localization as α-SMA expression appears to disrupt fibroblast projection alignment with collagen in the peripheral sclera, but not the ppScl, and fibroblast orientation is most altered when cells detect both strain and TGF-β signaling simultaneously [ 112 ]. These synergistic processes likely reinforce chronic glaucomatous remodeling of the ppScl, altering its biomechanical properties.…”