Immune Responses Induced at One Hour Post Cataract Surgery Wounding of the Chick Lens.

While the lens is an avascular tissue with an immune-privileged status, studies have now revealed that there are immune responses specifically linked to the lens. The response to lens injury, such as following cataract surgery, has been shown to involve the activation of the resident immune cell population of the lens and the induction of immunomodulatory factors by the wounded epithelium. However, there has been limited investigation into the immediate response of the lens to wounding, particularly those induced factors that are intrinsic to the lens and its associated resident immune cells. Using an established chick embryo ex vivo cataract surgery model has made it possible to determine the early immune responses of this tissue to injury, including its resident immune cells, through a transcriptome analysis. RNA-seq studies were performed to determine the gene expression profile at 1 h post wounding compared to time 0. The results provided evidence that, as occurs in other tissues, the resident immune cells of the lens rapidly acquired a molecular signature consistent with their activation. These studies also identified the expression of many inflammatory factors by the injured lens that are associated with both the induction and regulation of the immune response.

The involvement of caspases in the process of nuclear removal during lens fiber cell differentiation.

The terminal differentiation of lens fiber cells involves elimination of their organelles, which must occur while still maintaining their functionality throughout a lifetime. Removal of non-nuclear organelles is accomplished through induction of autophagy following the spatiotemporal suppression of the PI3K/Akt signaling axis. However, blocking this pathway is not alone sufficient to induce removal of fiber cell nuclei. While the final steps in fiber cell nuclear elimination are highlighted by the appearance of TUNEL-positive nuclei, which are associated with activation of the lens-specific DNaseIIß, there are many steps in the process that precede the appearance of double stranded DNA breaks. We showed that this carefully regulated process, including the early changes in nuclear morphology resulting in nuclear condensation, cleavage of lamin B, and labeling by pH2AX, is reminiscent of the apoptotic process associated with caspase activation. Multiple caspases are known to be expressed and activated during lens cell differentiation. In this study, we investigated the link between two caspase downstream targets associated with apoptosis, ICAD, whose cleavage by caspase-3 leads to activation of CAD, a DNase that can create both single- and double-stranded DNA cleavages, and lamin B, a primary component of the nuclear lamina. We discovered that the specific inhibition of caspase-3 activation prevents both lamin B and DNA cleavage. Inhibiting caspase-3 did not prevent nuclear condensation or removal of the nuclear membrane. In contrast, a pan-caspase inhibitor effectively suppressed condensation of fiber cell nuclei during differentiation. These studies provide evidence that caspases play an important role in the process of removing fiber cell nuclei during lens differentiation.

Examining PI3K-signaling-dependent regulation of lens organelle free zone formation via immunolocalization and immunoblotting in chick embryos.

The elimination of lens organelles during development, required for mature lens function, is an autophagy-dependent mechanism induced through suppression of PI3K signaling. Here, we present a protocol for investigating the signaling pathways responsible for induction of the formation of this lens organelle free zone. We describe steps for preparation of lens organ culture and use of signaling pathway inhibitors. We then detail procedures for analyzing their impact using both confocal microscopy imaging of immunolabeled lens cryosections and immunoblot approaches. For complete details on the use and execution of this protocol, please refer to Gheyas et al. (2022).1.

Multiomic analysis implicates FOXO4 in genetic regulation of chick lens fiber cell differentiation.

A classic model for identification of novel differentiation mechanisms and pathways is the eye lens that consists of a monolayer of quiescent epithelial cells that are the progenitors of a core of mature fully differentiated fiber cells. The differentiation of lens epithelial cells into fiber cells follows a coordinated program involving cell cycle exit, expression of key structural proteins and the hallmark elimination of organelles to achieve transparency. Although multiple mechanisms and pathways have been identified to play key roles in lens differentiation, the entirety of mechanisms governing lens differentiation remain to be discovered. A previous study established that specific chromatin accessibility changes were directly associated with the expression of essential lens fiber cell genes, suggesting that the activity of transcription factors needed for expression of these genes could be regulated through binding access to the identified chromatin regions. Sequence analysis of the identified chromatin accessible regions revealed enhanced representation of the binding sequence for the transcription factor FOXO4 suggesting a direct role for FOXO4 in expression of these genes. FOXO4 is known to regulate a variety of cellular processes including cellular response to metabolic and oxidative stress, cell cycle withdrawal, and homeostasis, suggesting a previously unidentified role for FOXO4 in the regulation of lens cell differentiation. To further evaluate the role of FOXO4 we employed a multiomics approach to analyze the relationship between genome-wide FOXO4 binding, the differentiation-specific expression of key genes, and chromatin accessibility. To better identify active promoters and enhancers we also examined histone modification through analysis of H3K27ac. Specific methods included CUT&RUN (FOXO4 binding and H3K27ac modification), RNA-seq (differentiation state specific gene expression), and ATAC-seq (chromatin accessibility). CUT&RUN identified 20,966 FOXO4 binding sites and 33,921 H3K27ac marked regions across the lens fiber cell genome. RNA-seq identified 956 genes with significantly greater expression levels in fiber cells compared to epithelial cells (log2FC > 0.7, q < 0.05) and 2548 genes with significantly lower expression levels (log2FC < -0.7, q < 0.05). Integrated analysis identified 1727 differentiation-state specific genes that were nearest neighbors to at least one FOXO4 binding site, including genes encoding lens gap junctions (GJA1, GJA3), lens structural proteins (BFSP1, CRYBB1, ASL1), and genes required for lens transparency (HSF4, NRCAM). Multiomics analysis comparing the identified FOXO4 binding sites in published ATAC-seq data revealed that chromatin accessibility was associated with FOXO4-dependent gene expression during lens differentiation. The results provide evidence for an important requirement for FOXO4 in the regulated expression of key genes required for lens differentiation and link epigenetic regulation of chromatin accessibility and H3K27ac histone modification with the function of FOXO4 in controlling lens gene expression during lens fiber cell differentiation.

Immune Cells Localize to Sites of Corneal Erosions in C57BL/6 Mice.

Recurrent epithelial erosions develop in the cornea due to prior injury or genetic predisposition. Studies of recurrent erosions in animal models allow us to gain insight into how erosions form and are resolved. While slowing corneal epithelial cell migration and reducing their proliferation following treatment with mitomycin C reduce erosion formation in mice after sterile debridement injury, additional factors have been identified related to cytokine expression and immune cell activation. The relationship between recruitment of immune cells to the region of the cornea where erosions form and their potential roles in erosion formation and/or erosion repair remains unexplored in the C57BL/6 mouse recurrent erosion model. Here, high resolution imaging of mouse corneas was performed at D1, D7, and D28 after dulled-blade debridement injury in C57BL/6 mice. Around 50% of these mice have frank corneal erosions at D28 after wounding. A detailed assessment of corneas revealed the involvement of M2 macrophages in both frank and developing erosions at early stages of their formation.

Autophagy Requirements for Eye Lens Differentiation and Transparency.

Recent evidence points to autophagy as an essential cellular requirement for achieving the mature structure, homeostasis, and transparency of the lens. Collective evidence from multiple laboratories using chick, mouse, primate, and human model systems provides evidence that classic autophagy structures, ranging from double-membrane autophagosomes to single-membrane autolysosomes, are found throughout the lens in both undifferentiated lens epithelial cells and maturing lens fiber cells. Recently, key autophagy signaling pathways have been identified to initiate critical steps in the lens differentiation program, including the elimination of organelles to form the core lens organelle-free zone. Other recent studies using ex vivo lens culture demonstrate that the low oxygen environment of the lens drives HIF1a-induced autophagy via upregulation of essential mitophagy components to direct the specific elimination of the mitochondria, endoplasmic reticulum, and Golgi apparatus during lens fiber cell differentiation. Pioneering studies on the structural requirements for the elimination of nuclei during lens differentiation reveal the presence of an entirely novel structure associated with degrading lens nuclei termed the nuclear excisosome. Considerable evidence also indicates that autophagy is a requirement for lens homeostasis, differentiation, and transparency, since the mutation of key autophagy proteins results in human cataract formation.

PI3K Isoform-Specific Regulation of Leader and Follower Cell Function for Collective Migration and Proliferation in Response to Injury.

To ensure proper wound healing it is important to elucidate the signaling cues that coordinate leader and follower cell behavior to promote collective migration and proliferation for wound healing in response to injury. Using an ex vivo post-cataract surgery wound healing model we investigated the role of class I phosphatidylinositol-3-kinase (PI3K) isoforms in this process. Our findings revealed a specific role for p110α signaling independent of Akt for promoting the collective migration and proliferation of the epithelium for wound closure. In addition, we found an important role for p110α signaling in orchestrating proper polarized cytoskeletal organization within both leader and wounded epithelial follower cells to coordinate their function for wound healing. p110α was necessary to signal the formation and persistence of vimentin rich-lamellipodia extensions by leader cells and the reorganization of actomyosin into stress fibers along the basal domains of the wounded lens epithelial follower cells for movement. Together, our study reveals a critical role for p110α in the collective migration of an epithelium in response to wounding.

The Pro-Fibrotic Response to Lens Injury Is Signaled in a PI3K Isoform-Specific Manner.

The signaling inputs that function to integrate biochemical and mechanical cues from the extracellular environment to alter the wound-repair outcome to a fibrotic response remain poorly understood. Here, using a clinically relevant post-cataract surgery wound healing/fibrosis model, we investigated the role of Phosphoinositide-3-kinase (PI3K) class I isoforms as potential signaling integrators to promote the proliferation, emergence and persistence of collagen I-producing alpha smooth muscle actin (αSMA+) myofibroblasts that cause organ fibrosis. Using PI3K isoform specific small molecule inhibitors, our studies revealed a requisite role for PI3K p110α in signaling the CD44+ mesenchymal leader cell population that we previously identified as resident immune cells to produce and organize a fibronectin-EDA rich provisional matrix and transition to collagen I-producing αSMA+ myofibroblasts. While the PI3K effector Akt was alone insufficient to regulate myofibroblast differentiation, our studies revealed a role for Rac, another potential PI3K effector, in this process. Our studies further uncovered a critical role for PI3K p110α in signaling the proliferation of CD44+ leader cells, which is important to the emergence and expansion of myofibroblasts. Thus, these studies identify activation of PI3K p110α as a critical signaling input following wounding to the development and progression of fibrotic disease.

The Pro-fibrotic Response of Mesenchymal Leader Cells to Lens Wounding Involves Hyaluronic Acid, Its Receptor RHAMM, and Vimentin.

Hyaluronic Acid/Hyaluronan (HA) is a major component of the provisional matrix deposited by cells post-wounding with roles both in regulating cell migration to repair a wound and in promoting a fibrotic outcome to wounding. Both are mediated through its receptors CD44 and RHAMM. We now showed that HA is present in the provisional matrix assembled on the substrate surface in a lens post-cataract surgery explant wound model in which mesenchymal leader cells populate the wound edges to direct migration of the lens epithelium across the adjacent culture substrate onto which this matrix is assembled. Inhibiting HA expression with 4-MU blocked assembly of FN-EDA and collagen I by the wound-responsive mesenchymal leader cells and their migration. These cells express both the HA receptors CD44 and RHAMM. CD44 co-localized with HA at their cell-cell interfaces. RHAMM was predominant in the lamellipodial protrusions extended by the mesenchymal cells at the leading edge, and along HA fibrils organized on the substrate surface. Within a few days post-lens wounding the leader cells are induced to transition to αSMA+ myofibroblasts. Since HA/RHAMM is implicated in both cell migration and inducing fibrosis we examined the impact of blocking HA synthesis on myofibroblast emergence and discovered that it was dependent on HA. While RHAMM has not been previously linked to the intermediate filament protein vimentin, our studies with these explant cultures have shown that vimentin in the cells' lamellipodial protrusions regulate their transition to myofibroblast. PLA studies now revealed that RHAMM was complexed with both HA and vimentin in the lamellipodial protrusions of leader cells, implicating this HA/RHAMM/vimentin complex in the regulation of leader cell function post-wounding, both in promoting cell migration and in the transition of these cells to myofibroblasts. These results increase our understanding of how the post-wounding matrix environment interacts with receptor/cytoskeletal complexes to determine whether injury outcomes are regenerative or fibrotic.

Patterns of Crystallin Gene Expression in Differentiation State Specific Regions of the Embryonic Chicken Lens.

Purpose: Transition from lens epithelial cells to lens fiber cell is accompanied by numerous changes in gene expression critical for lens transparency. We identify expression patterns of highly prevalent genes including ubiquitous and enzyme crystallins in the embryonic day 13 chicken lens. Methods: Embryonic day 13 chicken lenses were dissected into central epithelial cell (EC), equatorial epithelial cell (EQ), cortical fiber cell (FP), and nuclear fiber cell (FC) compartments. Total RNA was prepared, subjected to high-throughput unidirectional mRNA sequencing, analyzed, mapped to the chicken genome, and functionally grouped. Results: A total of 77,097 gene-specific transcripts covering 17,450 genes were expressed, of which 10,345 differed between two or more lens subregions. Ubiquitous crystallin gene expression increased from EC to EQ and was similar in FP and FC. Highly expressed crystallin genes fell into three coordinately expressed groups with R2 ≥ 0.93: CRYAA, CRYBB2, CRYAB, and CRYBA2; CRYBB1, CRYBA4, CRYGN, ASL1, and ASL; and CRYBB3 and CRYBA1. The highly expressed transcription factors YBX1, YBX3, PNRC1, and BASP1 were coordinately expressed with the second group of crystallins (r2 > 0.88). Conclusions: Although it is well known that lens crystallin gene expression changes during the epithelial to fiber cell transition, these data identify for the first time three distinct patterns of expression for specific subsets of crystallin genes, each highly correlated with expression of specific transcription factors. The results provide a quantitative basis for designing functional experiments pinpointing the mechanisms governing the landscape of crystallin expression during fiber cell differentiation to attain lens transparency.

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone.

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.

Uveitis-mediated immune cell invasion through the extracellular matrix of the lens capsule.

While the eye is considered an immune privileged site, its privilege is abrogated when immune cells are recruited from the surrounding vasculature in response to trauma, infection, aging, and autoimmune diseases like uveitis. Here, we investigate whether in uveitis immune cells become associated with the lens capsule and compromise its privilege in studies of C57BL/6J mice with experimental autoimmune uveitis. These studies show that at D14, the peak of uveitis in these mice, T cells, macrophages, and Ly6G/Ly6C+ immune cells associate with the lens basement membrane capsule, burrow into the capsule matrix, and remain integrated with the capsule as immune resolution is occurring at D26. 3D surface rendering image analytics of confocal z-stacks and scanning electron microscopy imaging of the lens surface show the degradation of the lens capsule as these lens-associated immune cells integrate with and invade the lens capsule, with a subset infiltrating both epithelial and fiber cell regions of lens tissue, abrogating its immune privilege. Those immune cells that remain on the surface often become entwined with a fibrillar net-like structure. Immune cell invasion of the lens capsule in uveitis has not been described previously and may play a role in induction of lens and other eye pathologies associated with autoimmunity.

The ciliary zonules provide a pathway for immune cells to populate the avascular lens during eye development.

The eye is an immune-privileged site, with both vasculature and lymphatics absent from the central light path. Unique adaptations have made it possible for immune cells to be recruited to this region of the eye in response to ocular injuries and pathogenic insults. The induction of such immune responses is typically activated by tissue resident immune cells, considered the sentinels of the immune system. We discovered that, despite the absence of an embedded vasculature, the embryonic lens becomes populated by resident immune cells. The paths by which they travel to the lens during development were not known. However, our previous studies show that in response to corneal wounding immune cells travel to the lens from the vascular-rich ciliary body across the zonules that link these two tissues. We now examined whether the zonule fibers provide a path for immune cells to the embryonic lens, and the zonule-associated matrix molecules that could promote immune cell migration. The vitreous also was examined as a potential source of lens resident immune cells. This matrix-rich site in the posterior of the eye harbors hyalocytes, an immune cell type with macrophage-like properties. We found that both the zonules and the vitreous of the embryonic eye contained fibrillin-2-based networks and that migration-promoting matrix proteins like fibronectin and tenascin-C were linked to these fibrils. Immune cells were seen emerging from the ciliary body, migrating along the ciliary zonules to the lens, and invading through the lens capsule at its equator. This is just adjacent to where immune cells take up residence in the embryonic lens. In contrast, the immune cells of the vitreous were not detected in the region of the lens. These results strongly suggest that the ciliary zonules are a primary path of immune cell delivery to the developing lens.

Descemet's membrane injury and regeneration, and posterior corneal fibrosis, in rabbits.

The purpose of this investigation was to study Descemet's membrane and corneal endothelial regeneration, myofibroblast generation and disappearance, and TGF beta-1 localization after Descemet's membrane-endothelial excision (Descemetorhexis) in rabbits. Thirty-six rabbits had 8 mm Descemetorhexis and standardized slit lamp photos at 1, 2 and 4 days, 1, 2 and 4 weeks, and 2, 4 and 6 months, as well as multiplex IHC for stromal cell markers keratocan, vimentin, and alpha-smooth muscle actin (SMA); basement membrane (BM) components perlecan, nidogen-1, laminin alpha-5, and collagen type IV; and corneal endothelial marker Na,K-ATPase ß1, and TGF beta-1, with ImageJ quantitation. Stromal transparency increased from the periphery beginning at two months after injury and progressed into the central cornea by six months. At six months, central transparency was primarily limited by persistent mid-stromal neovascularization. Stromal myofibroblast zone thickness in the posterior stroma peaked at one month after injury, and then progressively decreased until to six months when few myofibroblasts remained. The regeneration of a laminin alpha-5 and nidogen-1 Descemet's membrane "railroad track" structure was accompanied by corneal endothelial closure and stromal cell production of BM components in corneas from four to six months after injury. TGF beta-1 deposition at the posterior corneal surface from the aqueous humor peaked at one day after Descemetorhexis and diminished even before regeneration of the endothelium and Descemet's membrane. This decrease was associated with collagen type IV protein production by corneal fibroblasts, and possibly myofibroblasts, in the posterior stroma. Descemet's membrane and the corneal endothelium regenerated in the rabbit cornea by six months after eight mm Descemetorhexis. Real-time quantitative RT-PCR experiments in vitro with marker-verified rabbit corneal cells found that 5 ng/ml or 10 ng/ml TGF beta-1 upregulated col4a1 or col4a2 mRNA expression after 6 h or 12 h of exposure in corneal fibroblasts, but not in myofibroblasts. Stromal cells produced large amounts of collagen type IV that likely decreased TGF beta-1 penetration into the stroma and facilitated the resolution of myofibroblast-generated fibrosis.

Immune cells in lens injury repair and fibrosis.

Immune cells, both tissue resident immune cells and those immune cells recruited in response to wounding or degenerative conditions, are essential to both the maintenance and restoration of homeostasis in most tissues. These cells are typically provided to tissues by their closely associated vasculatures. However, the lens, like many of the tissues in the eye, are considered immune privileged sites because they have no associated vasculature. Such absence of immune cells was thought to protect the lens from inflammatory responses that would bring with them the danger of causing vision impairing opacities. However, it has now been shown, as occurs in other immune privileged sites in the eye, that novel pathways exist by which immune cells come to associate with the lens to protect it, maintain its homeostasis, and function in its regenerative repair. Here we review the discoveries that have revealed there are both innate and adaptive immune system responses to lens, and that, like most other tissues, the lens harbors a population of resident immune cells, which are the sentinels of danger or injury to a tissue. While resident and recruited immune cells are essential elements of lens homeostasis and repair, they also become the agents of disease, particularly as progenitors of pro-fibrogenic myofibroblasts. There still remains much to learn about the function of lens-associated immune cells in protection, repair and disease, the knowledge of which will provide new tools for maintaining the core functions of the lens in the visual system.

Immune responses to injury and their links to eye disease.

The eye is regarded as an immune privileged site. Since the presence of a vasculature would impair vision, the vasculature of the eye is located outside of the central light path. As a result, many regions of the eye evolved mechanisms to deliver immune cells to sites of dysgenesis, injury, or in response to the many age-related pathologies. While the purpose of these immune responses is reparative or protective, cytokines released by immune cells compromise visual acuity by inducing inflammation and fibrosis. The response to traumatic or pathological injury is distinct in different regions of the eye. Age-related diseases impact both the anterior and posterior segment and lead to reduced quality of life and blindness. Here we focus attention on the role that inflammation and fibrosis play in the progression of age-related pathologies of the cornea and the lens as well as in glaucoma, the formation of epiretinal membranes, and in proliferative vitreoretinopathy.

Resident immune cells of the avascular lens: Mediators of the injury and fibrotic response of the lens.

Tissues typically harbor subpopulations of resident immune cells that function as rapid responders to injury and whose activation leads to induction of an adaptive immune response, playing important roles in repair and protection. Since the lens is an avascular tissue, it was presumed that it was absent of resident immune cells. Our studies now show that resident immune cells are a shared feature of the human, mouse, and chicken lens epithelium. These resident immune cells function as immediate responders to injury and rapidly populate the wound edge following mock cataract surgery to function as leader cells. Many of these resident immune cells also express MHCII providing them with antigen presenting ability to engage an adaptive immune response. We provide evidence that during development immune cells migrate on the ciliary zonules and localize among the equatorial epithelial cells of the lens adjacent to where the ciliary zonules associate with the lens capsule. These findings suggest that the vasculature-rich ciliary body is a source of lens resident immune cells. We identified a major role for these cells as rapid responders to wounding, quickly populating each wound were they can function as leaders of lens tissue repair. Our findings also show that lens resident immune cells are progenitors of myofibroblasts, which characteristically appear in response to lens cataract surgery injury, and therefore, are likely agents of lens pathologies to impair vision like fibrosis.

Specification of the patterning of a ductal tree during branching morphogenesis of the submandibular gland.

The development of ductal structures during branching morphogenesis relies on signals that specify ductal progenitors to set up a pattern for the ductal network. Here, we identify cellular asymmetries defined by the F-actin cytoskeleton and the cell adhesion protein ZO-1 as the earliest determinants of duct specification in the embryonic submandibular gland (SMG). Apical polarity protein aPKCζ is then recruited to the sites of asymmetry in a ZO-1-dependent manner and collaborates with ROCK signaling to set up apical-basal polarity of ductal progenitors and further define the path of duct specification. Moreover, the motor protein myosin IIB, a mediator of mechanical force transmission along actin filaments, becomes localized to vertices linking the apical domains of multiple ductal epithelial cells during the formation of ductal lumens and drives duct maturation. These studies identify cytoskeletal, junctional and polarity proteins as the early determinants of duct specification and the patterning of a ductal tree during branching morphogenesis of the SMG.

Dynamics of the lens basement membrane capsule and its interaction with connective tissue-like extracapsular matrix proteins.

The lens, suspended in the middle of the eye by tendon-like ciliary zonule fibers and facing three different compartments of the eye, is enclosed in what has been described as the thickest basement membrane in the body. While the protein components of the capsule have been a subject of study for many years, the dynamics of capsule formation, and the region-specific relationship of its basement membrane components to one another as well as to other matrix molecules remains to be explored. Through high resolution confocal and super-resolution imaging of the lens capsule and 3D surface renderings of acquired z-stacks, our studies revealed that each of its basement membrane proteins, laminin, collagen IV, nidogen and perlecan, has unique structure, organization, and distribution specific both to the region of the lens that the capsule is located in and the position of the capsule within the eye. We provide evidence of basal membrane gradients across the depth of the capsule as well as the synthesis of distinct basement membrane lamella within the capsule. These distinctions are most prominent in the equatorial capsule zone where collagen IV and nidogen span the capsule depth, while laminin and perlecan are located in two separate lamellae located at the innermost and outermost capsule domains. We discovered that an extracapsular matrix compartment rich in the connective tissue-like matrix molecules fibronectin, tenascin-C, and fibrillin is integrated with the superficial surface of the lens capsule. Each matrix protein in this extracapsular zone also exhibits region-specific distribution with fibrils of fibrillin, the matrix protein that forms the backbone of the ciliary zonules, inserting within the laminin/perlecan lamella at the surface of the equatorial lens capsule.

TGFß1 and TGFß2 proteins in corneas with and without stromal fibrosis: Delayed regeneration of apical epithelial growth factor barrier and the epithelial basement membrane in corneas with stromal fibrosis.

The purpose of this study was to investigate the expression and localization of transforming growth factor (TGF) ß1 and TGFß2 in rabbit corneas that healed with and without stromal fibrosis, and to further study defective perlecan incorporation in the epithelial basement membrane (EBM) in corneas with scarring fibrosis. A total of 120 female rabbits had no surgery, -4.5D PRK, or -9D PRK. Immunohistochemistry (IHC) was performed at time points from unwounded to eight weeks after surgery, with four corneas at each time point in each group. Multiplex IHC was performed for TGFß1 or TGFß2, with Image-J quantitation, and keratocan, vimentin, alpha-smooth muscle actin (SMA), perlecan, laminin-alpha 5, nidogen-1 or CD11b. Corneas at the four-week peak for myofibroblast and fibrosis development were evaluated using Imaris 3D analysis. Delayed regeneration of both an apical epithelial growth factor barrier and EBM barrier function, including defective EBM perlecan incorporation, was greater in high injury -9D PRK corneas compared to -4.5D PRK corneas without fibrosis. Defective apical epithelial growth factor barrier and EBM allowed epithelial and tear TGFß1 and tear TGFß2 to enter the corneal stroma to drive myofibroblast generation in the anterior stroma from vimentin-positive corneal fibroblasts, and likely fibrocytes. Vimentin-positive cells and unidentified vimentin-negative, CD11b-negative cells also produce TGFß1 and/or TGFß2 in the stroma in some corneas. TGFß1 and TGFß2 were at higher levels in the anterior stroma in the weeks preceding myofibroblast development in the -9D group. All -9D corneas (beginning two to three weeks after surgery), and four -4.5D PRK corneas developed significant SMA + myofibroblasts and stromal fibrosis. Both the apical epithelial growth factor barrier and/or EBM barrier functions tended to regenerate weeks earlier in -4.5D PRK corneas without fibrosis, compared to -4.5D or -9D PRK corneas with fibrosis. SMA-positive myofibroblasts were markedly reduced in most corneas by eight weeks after surgery. The apical epithelial growth factor barrier and EBM barrier limit TGFß1 and TGFß2 entry into the corneal stroma to modulate corneal fibroblast and myofibroblast development associated with scarring stromal fibrosis. Delayed regeneration of these barriers in corneas with more severe injuries promotes myofibroblast development, prolongs myofibroblast viability and triggers stromal scarring fibrosis.