Latent-transforming growth factor beta-binding protein-2 (LTBP-2) is required for longevity but not for development of zonular fibers

Abstract

Latent-transforming growth factor beta-binding protein 2 (LTBP-2) is a major component of arterial and lung tissue and of the ciliary zonule, the system of extracellular fibers that centers and suspends the lens in the eye. LTBP-2 has been implicated previously in the development of extracellular microfibrils, although its exact role remains unclear. Here, we analyzed the three-dimensional structure of the ciliary zonule in wild type mice and used a knockout model to test the contribution of LTBP-2 to zonule structure and mechanical properties. In wild types, zonular fibers had diameters of 0.5-1.0 micrometers, with an outer layer of fibrillin-1-rich microfibrils and a core of fibrillin-2-rich microfibrils. LTBP-2 was present in both layers. The absence of LTBP-2 did not affect the number of fibers, their diameters, nor their coaxial organization. However, by two months of age, LTBP-2-depleted fibers began to rupture, and by six months, a fully penetrant ectopia lentis phenotype was present, as confirmed by in vivo imaging. To determine whether the seemingly normal fibers of young mice were compromised mechanically, we compared zonule stress/strain relationships of wild type and LTBP-2-deficient mice and developed a quasi-linear viscoelastic engineering model to analyze the resulting data. In the absence of LTBP-2, the ultimate tensile strength of the zonule was reduced by about 50%, and the viscoelastic behavior of the fibers was altered significantly. We developed a harmonic oscillator model to calculate the forces generated during saccadic eye movement. Model simulations suggested that mutant fibers are prone to failure during rapid rotation of the eyeball. Together, these data indicate that LTBP-2 is necessary for the strength and longevity of zonular fibers, but not necessarily for their formation.

Keywords: Ectopia lentis; LTBP-2; Microspherophakia; Quasi-linear viscoelastic model; Saccade; Super-resolution microscopy; Zonule.

Figure 1.

Arrangement of the lens and ciliary zonule in a one-month-old mouse eye, drawn approximately to scale. A. Organization of the anterior segment, showing the relationship between lens, zonule, and wall of the eye. B. The zonule extends from the posterior portion of the ciliary body (CB, blue) to the lens. Zonular fibers diverge as they approach the lens. The population of zonular fibers is arbitrarily divided into three groupings. Equatorial fibers (eq.) are the shortest fibers and are oriented normal to the equatorial lens surface. The anterior-most fibers (ant.) form an angle, θ₁, with the eq. fibers. Likewise, the posterior-most fibers (post.) form an angle, θ₂, with the eq. fibers. Total fiber divergence is θ₁ + θ₂. At the lens surface, the “zonular span” is the linear distance between the anchorage points of the anterior-most and posterior-most fibers (arrowheads).

Figure 2.

Three-dimensional reconstruction of wild type mouse zonule at 1 month of age, visualized by fibrillin-1 immunofluorescence. A. Maximum intensity orthographic projection of the zonule showing the anterior (ant.), equatorial (eq.), and posterior (post.) fibers as they approach the lens. The posterior fibers connect to the fibrillar girdle (FG) at the lens surface. B. XZ projections show the zonule from the lateral aspect (for orientation, see Fig. 1B). C. An individual optical section from 100 μm above the equatorial lens surface (position arrowed in B). D. An optical section from immediately above the lens surface (z = 0 μm, arrowed in B). Note the presence of channels (*1-3) in the zonular fibers. The channels meander but are generally oriented to the anterior-posterior axis. E. YZ projections show the channels in transverse section. They are approximately 30 μm wide and 50 μm high. F. Volumetric projection of individual zonular fibers showing their branched organization. Scale bars = 50 μm.

Figure 3.

Distribution of cross-sectional diameters of zonular fibers imaged 50 μm above the equatorial lens surface in 1-month-old wild type (upper panel) or Ltbp2-null mice (lower panel). The distributions are positively skewed, with a modal value in all cases of 0.55-0.60 μm. The posterior fiber distributions are the most heavily skewed, reflecting the presence of a higher proportion of thicker fibers in that region.

Figure 4.

Representative examples of empirical force measurements (black), and best fit of the QLV model for zonular force (red) and fiber failure rates (blue) for the ciliary zonule in one-month-old wild type and Ltbp2-null mice.

Figure 5.

Development of ectopia lentis in Ltbp2-null mice. A. OCT images of the anterior segment in age-matched wild type and Ltbp2-null mice. In this example, posterior lens displacement occurs at five months of age in the Ltbp2-null mouse (right hand column). Note that lens dislocation is accompanied by flattening of the iris. B. Variation of anterior chamber depth (ACD; see panel A) with age in eyes from wild type and Ltbp2-null mice as measured by OCT. Ectopia lentis is first apparent at 3 months of age and is fully penetrant by 6 months of age. C. Zonular dehiscence (area between the arrows) is observed as early as one month of age in some Ltbp2-null mice. D. By six months of age, the regions containing broken fibers have expanded significantly. E. In contrast, in six-month-old wild type mice, the zonular fibers are intact. Scale bar C-E = 500 μm.

Figure 6.

Displacement of lens relative to the limbus for 20 cycles of simulated saccadic motion (blue) and corresponding fraction of broken zonular fibers (orange). Zonular fibers in the wild type eye (left panel) resist the forces generated by oscillatory eye motion, while fibers in the Ltbp2-null eye (right panel) begin to break.

Figure 7.

Immunolocalization of fibrillin-1, fibrillin-2, and LTBP-2 in zonular fibers of one-month-old wild type and Ltbp2-null mice. A. TEM image of a wild type fiber in longitudinal section. The fiber is comprised of many microfibrils. Note the presence of faint cross-striations oriented perpendicular to the long axis of the fiber. B. Immuno-gold localization of LTBP-2 in a zonular fiber. LTBP-2 is distributed throughout the fiber. Inset. LTBP-2 labeling of individual microfibrils suggests a periodicity of about 200 nm. C,D. Transverse views of zonular fibers from wild type mice imaged using super-resolution microscopy. C. Fibrillin-2 (red) is present throughout the fiber but the staining is discontinuous and only partially overlaps with LTBP-2 (green) immunofluorescence, which is also discontinuous. D. Fibrillin-1 immunofluorescence (red) is restricted to a ≈200 nm-thick layer at the fiber surface and absent from the core. E,F. Transverse views of zonular fibers from Ltbp2-null mice. E. Fibrillin-2 immunofluorescence (red) is more homogeneous than in the wild type case (C). LTBP-2 immunofluorescence (green) is undetectable. F. As with wild type fibers, fibrillin-1 immunofluorescence (red) is restricted to the outer portion of the fiber. Scale bar inset B = 200 nm, all other panels are shown at the magnification indicated in F.

Figure 8.

LacZ expression in the eyes of Ltbp2-null mice. A, B. At postnatal day 5 (P5), LacZ expression is undetectable. C. At P7, weak LacZ expression (blue) is present in the non-pigmented ciliary epithelium (NPCE) and connective tissue of the eye wall. By P21, strong LacZ expression is evident in NPCE cells and the inner wall of Schlemm’s canal (SC). IE, iris epithelium, PCE, pigmented ciliary epithelium. Scale bar A = 250 μm, B-D =25 μm.

Figure 9.

Incorporation of LTBP-2 (green) into the nascent zonule. A. At P7, microfibrils (red, visualized with MAGP1/MFAP2 immunofluorescence) are associated with vessels of the tunica vasculosa lentis (TVL; arrowheads) and intervening regions of lens capsule. LTBP-2 is incorporated initially into circumferential fibers at the surface of the ciliary body (CB) and the proximal portions of radial fibers that connect the ciliary body to the lens equator. Nuclei (gray) are stained with DAPI, highlighting the meridional rows (MR) of lens epithelial cells underlying the zonular attachments to the lens capsule. B. By one month of age, LTBP-2 is present throughout the zonule, including the fibrillar girdle at the lens surface (FG). Scale bar = 50 μm.

Figure 10.

Zonular channels mark the original course of TVL capillaries at the lens surfaces. A. The zonule of a one-month-old wild type mouse is visualized using antibodies against fibrillin-1 (green) and MAGP-1 (light blue). Nuclei (red) are counterstained with a DNA probe. XZ projections show the zonular span and the presence of a capillary remnant (arrow). B. In XY projections, the nuclei of endothelial cells in the capillary (arrow) and equatorial lens cells are visible. The nuclei of differentiating lens fiber cells are aligned in meridional rows (MR). C. In XY projections, channels through the zonule are evident (*). The channels narrow posteriorly (arrowheads), forming ruts in the surface of the FG. Note that the immunofluorescence staining intensities for the two zonule proteins (fibrillin-1 and MAGP-1) are unequal; fibrillin-1 is present throughout the zonule, whereas MAGP-1 is enriched in the anterior and posterior fiber populations. D. Overlay of the fluorescent signals confirm that the capillaries lie entirely within the zonular channels. Scale bar = 50 μm.

Figure 11.

Schematic representation of the pull-up assay, showing geometric relationships between lens (blue), ciliary body (brown) and zonular fibers (red) in the unloaded (left) and loaded (right) states. In this assay, the eye is positioned “cornea down” and a lifting force (F) is applied to the back of the lens (blue). L_i and l_i = zonular fiber length; Z,z = position on z-axis; R,r = radius.

Figure 12.

Schematic of the harmonic oscillator model of rapid eye movement.

Figure 13.

Linear velocity of the limbus used to simulate a single rapid eye movement event. Angular velocity data were taken from Sakatani and Isa [19]. Eye sizes were measured elsewhere in this study (see Supplemental Data Table 1).