The mechanisms underlying ocular fibrotic diseases
Date
2017
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The normal function of the eye requires all key optical media (cornea, lens and retina) to be transparent. Disruption of any of these highly ordered tissues will lead to vision loss or even blindness. Many blinding conditions result from fibrosis, whose hallmarks are fibroblast to myofibroblast activation and proliferation, along with excessive extracellular matrix (ECM) synthesis and deposition. Many of the most common ocular diseases, such as corneal opacification, glaucoma, age-related macular degeneration (AMD), cataract, and diabetic retinopathy (DR), all have fibrotic features. Therefore, a better understanding of the mechanisms underlying ocular fibrotic diseases will contribute to the discovery of therapeutic approaches, which could potentially save patients’ vision acuity. ☐ In total, four studies are covered here, which includes β1-integrin deletion from the lens activates cellular stress responses leading to apoptosis and fibrosis (Chapter 3); Egr1’s role in Posterior Capsular Opacification (PCO), the fibrotic response of lens epithelial cells to cataract surgery (Chapter 4); the molecular mechanisms of Aniridia Fibrosis Syndrome (AFS) (Chapter 5); and the activation of canonical Wnt signaling during embryonic eye development and PCO (Chapter 6). Each chapter has a slightly different focus. Chapter 3 and part of Chapter 6 focused on β1-integrin and canonical Wnt signaling’s functionality during embryonic lens development and how alterations in these processes might result in fibrosis. Chapter 4 and the rest part of Chapter 6 elucidated Egr1 and canonical Wnt signaling’s role after cataract surgery, which could contribute to fibrotic PCO. In Chapter 5, a rare pan ocular fibrotic condition, AFS, was studied. All these will provide us a more solid understanding about how fibrosis occurs throughout the eye. ☐ β1-integrin deletion from the lens activates cellular stress responses leading to apoptosis and fibrosis. --
Previous research revealed that conditional deletion of β1-integrin from the mouse lens after E13.5 (β1MLR10) can lead to perinatal lens epithelial cell (LECs) apoptosis and microphthalmia in the adulthood. To elucidate the molecular connections between β1-integrin deletion and this phenotype, RNA-sequencing was performed. 120 differentially regulated genes (DRGs) were identified in β1MLR10 lenses at E15.5, many of which are involved in the cellular stress response, fibrosis, and/or apoptosis.
Through bioinformatics analysis and literature searching, Egr1 (early growth response 1) was selected as target gene, since (1) it was greatly elevated in the β1MLR10 lens (2) it can mediate cellular stress responses in various cell types, and (3) it was recognized as an upstream regulator of many other DRGs. Meanwhile, the activation status of FAK/Erk, TGF-β and Akt signaling were studied via western blot and immunohistochemistry, which revealed that pErk1/2 and pAkt are elevated in β1MLR10 LECs. To test Egr1’s functionality in β1MLR10 lenses, mice lacking both β1-integrin and Egr1 genes from the lenses were created (β1MLR10/Egr1-/-), which confirmed that deletion of Egr1 from β1MLR10 lenses could partially rescue the microphthalmia phenotype.
These results provided the molecular mechanism underlying the microphthalmia observed in β1MLR10 mice. β1-integrin regulates the appropriate levels of Erk1/2 and Akt phosphorylation in LECs while its deficiency results in the overexpression of Egr1 and elevated cell death. ☐ The initial mechanism of fibrosis upon cataract surgery, Egr1’s role in Posterior Capsular Opacification (PCO) --
PCO is the most common side effect after cataract surgery, but its full mechanism, especially the initial responses occurring immediately after the surgery, and how they trigger later fibrotic responses remain unclear.
As one of the immediate early genes, Egr1 can be activated transiently and rapidly in response to cellular stimuli. It is involved in lens physiology and pathology, and the pathogenesis of multiple fibrotic diseases. More importantly, Egr1 regulates the expression of multiple fibrotic genes involved in TGF-β-dependent profibrotic responses, which is known to regulate PCO. Thus, Egr1 has the potential to mediate the initial responses during PCO.
In WT mice, Egr1 started to upregulate 3 hours post-surgery, while remaining detectable until 48h post-surgery. Its downstream negative regulator, Nab2, was also upregulated, ranging from 1h to 96 post-surgery. In Egr1-/- mice, the fibrotic response was attenuated 48h post-surgery, as fibrotic markers, CD44, αV-integrin and αSMA failed to upregulate. This phenomenon became less obvious/non-existent by 5 days post-surgery, since late stage fibrosis and fiber cell differentiation remained intact in Egr1-/- mice, with the upregulation of αSMA, Tenascin-C and cMaf. These results indicated that Egr1 could be a possible profibrotic transcription factor that functions at early times post cataract surgery.
By evaluating the levels of pSMAD3 and p300 at 48h and 5 days post-surgery, I confirmed that this unique fibrosis rebound was not due to changes in SMAD-dependent nor SMAD-independent TGF-β signaling. Instead, Ras-MEK1/2-Erk1/2 signaling and MMPs might be the key player involved. More in-depth future research is needed to elucidate the possible mechanisms involved. ☐ Molecular mechanisms of Aniridia Fibrosis Syndrome (AFS) --
Congenital aniridia (CI) is defined as iris hypoplasia at birth, and often results from PAX6 mutations/deletions. Surgical interventions to treat common CI sequelae can lead to devastating fibrotic complications (AFS). While little is known about the pathogenesis of AFS, previous studies showed that Pax6 negatively regulates canonical Wnt signaling in the embryonic eye. As canonical Wnt and TGF-β signaling can synergize to drive fibrosis, this work tests the hypothesis that Pax6 haploinsufficiency leads to upregulated Wnt signaling in the adult eye, which synergizes with surgically mediated TGF-β activation and results in AFS.
Both human AFS membranes and Pax6+/tm1Pgr full thickness central cornea wound (FTCCW) mouse model was used in the study. Human AFS tissues were comprised of α-smooth muscle actin (αSMA), pSMAD3 and nuclear β-catenin positive myofibroblasts embedded in fibrotic extracellular matrix. Unoperated adult Pax6+/tm1Pgr mice developed pockets of spontaneous fibrosis in both lens epithelial cells (LECs) and cornea stroma. Then, FTCCW was performed on WT and Pax6+/tm1Pgr mice to model post-surgery fibrotic responses. After surgery, Pax6+/tm1Pgr mice developed severe fibrosis at both the injury site and iris root associated with elevated levels of nuclear β-catenin and pSMAD3. They also exhibited elevated proliferation (Ki67) at the central cornea injury site. However, the expression of inflammation (Ly-6G/6C, CD11b, F4/80,) angiogenesis (Pecam), and apoptosis (cleaved caspase-3) markers were comparable between two groups.
To better study the activation of canonical Wnt signaling, Wnt reporter mice (TCF/Lef:H2B/GFP) were mated with Pax6+/tm1Pgr mice, to generate reporter carriers (Pax6+/tm1Pgr, R+). Compared to WT, unoperated Pax6+/tm1Pgr, R+ mice exhibited elevated canonical Wnt signaling in LECs as measured by GFP expression. After FTCCW, GFP and pSMAD3 found to be co-localized in SMA expressing cells at the injury site. However, Pax6+/tm1Pgr, R+ mice did not exhibit elevated fibrosis after injury, which is consistent with the known effects of genetic background on the phenotype of the aniridic eye.
In conclusion, Pax6 haploinsufficiency results in a propensity to develop spontaneous ocular fibrosis. As these eyes also exhibit increased canonical Wnt signaling activity, this may synergize with elevated TGF-β signaling induced upon surgery, to drive the pathogenesis of AFS. ☐ The activation of canonical Wnt signaling during embryonic eye development and posterior capsular opacification (PCO) --
Canonical Wnt signaling plays an important role during eye development, suppressing ectopic lens formation in the periocular ectoderm, and drives the differentiation of neural retinal precursor cells. However, it is difficult to evaluate canonical Wnt signaling at the cellular level. Since its downstream mediator, β-catenin, has dual intracellular functions, which is associated with both cadherin adhesion complex and gene transcription. Usage of Wnt reporter mice helps to differentiate β-catenin from its two functionality pools. Former canonical Wnt reporter mice that utilized genes, such as LacZ, were unable to indicate cell specific activation.
In this study, a new Wnt reporter mouse strain was used. It can reveal canonical Wnt activation on a single cell basis, by placing TCF/Lef responsive element and an hsp68 minimal promoter in front of green fluorescent protein (GFP). By visualizing GFP expression, I was able to reevaluate the extent of canonical (TCF/Lef mediated) Wnt signaling during embryonic eye development, and also test the hypothesis that this pathway is strongly activated during the formation of PCO. Embryos and postnatal mice heterozygous for the reporter gene were obtained from timed matings. Mouse cataract surgery was conducted in reporter carriers to collect post-surgery samples. Afterwards, GFP expression was analyzed by immunofluorescence staining and confocal microscopy.
Starting from E12.5, neural crest-derived periocular mesenchymal cells gradually migrate into the space between the lens vesicle and corneal epithelium, forming the corneal endothelium and stroma, with limited GFP signals detected. At E14.5, robust GFP expression was found in the putative cornea stroma and endothelium. At E16.5, the cornea is completely separated from the lens, while GFP expression also greatly decreased in the stroma, with some signals maintained in the endothelial cells. A similar pattern was found in P0 samples. Along with cornea stratification, GFP signals continued to decrease and became more confined. At P3, only a few GFP positive cells were found in the cornea endothelium. At P13, GFP signals shifted from endothelium to epithelium, which was completely gone in the stroma. At adulthood, abundant GFP expression was exclusively seen in the cornea epithelial cells.
In the retina, robust GFP expression was observed in the inner and outer neuroblastic layer at E14.5, which greatly decreased at E16.5. At P0, along with the retina stratification, robust GFP expression was found in the putative ganglion cells. At P3, strong GFP expression maintained in the ganglion cells, while signals in the inner nuclear and outer neuroblastic layer increased. At P13, both ganglion cells and inner nuclear layer showed extensive GFP expression, and the same pattern continued in the adult.
In the lens, no GFP expression was detected in either lens placode or lens pit. However, at E11.5, a few cells of the anterior lens vesicle were GFP positive. By E12.5, many cells of the newly formed lens epithelium exhibited GFP expression, and by E14.5, most cells of the lens epithelium were GFP positive. At later times in development, the GFP signal decreases, and in the normal adult lens, only occasional lens epithelial cells were GFP positive. However, after surgical fiber cell removal, GFP expression was highly upregulated in the mouse PCO model, along with robust αSMA expression.
All these suggested that the extent of canonical Wnt signaling is very dynamic in the developing eyes, which could upregulate dramatically upon surgically induced fibrosis in the lens. As canonical Wnt signaling can crosstalk with TGF-β signaling to drive fibrotic responses in other systems, these data suggest that canonical Wnt may synergize with TGF-β signaling to drive fibrotic PCO. ☐ Ocular fibrosis is a common pathological condition, which is featured in various ocular diseases. Depending on the context, the clinical manifestation could be very different. However, the underlying mechanisms have some shared features, especially the involvement of multiple signaling pathways. In this dissertation, four individual projects were included to elucidate the pathogenesis of ocular fibrosis from various angles, ranging from embryonic development to post-surgery fibrotic responses. Meanwhile, the in-depth study of some unique conditions, such as AFS and fibrotic responses after β1-integrin conditional deletion from the developing lens, lead to insightful understanding that could be applied broadly in other fibrotic conditions (i.e. PCO). All these findings will shed light on the comprehension of ocular fibrotic diseases, facilitating the exploration of potential clinical treatments and help patients enjoy better vision.
Description
Keywords
Biological sciences, Aniridia fibrosis syndrome (AFS), Beta1-integrin, Canonical wnt signaling, Egr1, Lens development, Posterior capsular opacification (PCO)