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PROGRAM | Biological Sciences

The Regulation of Transforming Growth Factor Beta (TGFβ) Signaling in Posterior Capsular Opacification

By: Mahbubul Shihan Chair: Melinda Duncan

ABSTRACT

Posterior capsular opacification (PCO), one of the major complications of cataract surgery, occurs when lens epithelial cells (LCs) left behind post cataract surgery (PCS) undergo epithelial to mesenchymal transition, migrate into the optical axis and produce opaque scar tissue. Despite preventive strategies such as modern cataract surgery and improved materials and shapes of intraocular lenses (IOLs), recent data suggests that about 28% of adults develop PCO at 5 years whereas 40% pediatric patients develop PCO by 2 years post cataract surgery (PCS).  The only FDA approved treatment for PCO is YAG laser capsulotomy which is not devoid of side effects. Besides, YAG laser can be unsuitable for pediatric patients, while the availability of YAG lasers and technical expertise are limited in developing and underdeveloped countries, suggesting that understanding the molecular mechanisms of PCO to develop preventive therapeutics would improve the outcome of cataract surgery. Although it is well established that activated transforming growth factor-beta (TGFβ) signaling mediates fibrotic PCO, the activation and bioavailability mechanisms of TGFβ signaling PCS are not well understood. Besides, if a preventive therapeutic against PCO is made available, at the start of my study, it was unclear whether clinicians treating cataract surgery patients would be interested in instituting it into their clinical practice.

In total, four studies are covered here. The first one is a survey-based study on understating cataract surgeons’ viewpoints on the clinical challenges they encounter in routine practice and the types of therapeutic interventions that would enhance the long-term efficacy of cataract surgery and PCO (Chapter 3). The next three studies are focused on understanding the molecular mechanisms of TGFβ signaling (the major mediator of PCO) regulation PCS. Chapter 4 focuses on understanding the ability of remnant LCs to express inflammatory cytokines leading to the infiltration of neutrophils and macrophages into the lens capsular bag, and the possible implications of these events in the activation of TGFβ signaling PCS. Chapter 5 focuses on identifying an αV integrin heterodimer that is critical for the activation of TGFβ signaling PCS and characterizes the effects of an antibody which can block integrin function PCS. Chapter 6 focuses on elucidating the regulatory role of a fibrotic extracellular matrix (ECM) molecule, fibronectin, in relationship to latent TGFβ complex regulation PCS. All these studies shed light on the molecular mechanisms of PCO development, and preventive therapeutic strategies to improve the outcome of cataract surgery and patient compliance.

 

Cataract surgeon viewpoints on the need for novel preventative anti-inflammatory and anti-posterior capsular opacification therapies

The purpose of this study was to determine cataract surgeon viewpoints on the efficacy of available therapies/preventatives for two common sequelae of cataract surgery: inflammation and posterior capsular opacification (PCO). To study this, cataract surgeons practicing worldwide specializing in adult, pediatric and veterinary patients were interviewed between March and August 2018. It was found that ocular inflammation PCS is treated by either corticosteroids and/or nonsteroidal anti-inflammatories (NSAIDs). Adult and pediatric cataract surgeons are satisfied with current treatments whereas this inflammation is still considered a problem by some in veterinary practice due to its slow resolution. Yttrium–aluminum–garnet (YAG) laser therapy is the PCO treatment of choice for adult cataract surgeons and they are generally pleased with its outcome. However, pediatric cataract surgeons find YAG problematic, especially in patients under 6 years of age, and invasive surgery is often needed to correct PCO/visual axis opacification (VAO). Veterinary ophthalmologists report that YAG is not effective for PCO in animals, especially dogs, due to the density of the fibrotic plaques; 86% of adults and 100% of veterinary and pediatric cataract surgeons surveyed agree that effective anti-PCO therapeutics would improve clinical care. In conclusion, surgeons treating human patients are pleased with the available treatments for ocular inflammation PCS, although some veterinary ophthalmologists disagree. The surgeons surveyed agree that PCO/VAO remains an unsolved problem in pediatric and veterinary cataract surgery while the long-term outcome of adult cataract surgery could be improved by additional attention to this issue.

Lens epithelial cells initiate an inflammatory response following cataract surgery

Lens epithelial cell (LC) conversion to myofibroblast is responsible for fibrotic cataract surgery complications including PCO. While TGFβ signaling is important, the mechanisms by which the TGFβ pathway is activated PCS are not well understood.

To understand this, RNA-seq was performed on LCs obtained from a mouse cataract surgery model at the time of surgery and 24 hours later. Bioinformatic analysis was performed with iPathwayGuide. Expression dynamics were determined by immunofluorescence. The findings suggest that the LC transcriptome is massively altered by 24 hours PCS. The differentially expressed genes included those important for lens biology and fibrotic markers. However, the most dramatic changes were in the expression of genes regulating the innate immune response, with the top three altered genes exhibiting greater than 1000-fold upregulation. Immunolocalization revealed that CXCL1, S100a9, CSF3, COX-2, CCL2, LCN2, and HMOX1 protein levels upregulate in LCs between 1 hour and 6 hours PCS and peak at 24 hours PCS, while their levels sharply attenuate by 3 days PCS. This massive upregulation of known inflammatory mediators precedes the infiltration of neutrophils into the eye at 18 hours PCS, the upregulation of canonical TGFβ signaling at 48 hours PCS, and the infiltration of macrophages at 3 days PCS. In conclusion, these data demonstrate that LCs produce proinflammatory cytokines immediately following lens injury that could drive postsurgical flare and suggest that inflammation may be a major player in the onset of lens-associated fibrotic disease PCS.

αVβ8 integrin- a potential druggable target to prevent posterior capsular opacification (PCO)

Previously, we found that αV integrins are critical in regulating TGFβ signaling mediated fibrotic PCO. Since each heterodimer has a different ligand binding profile/function and is inhibited by different compounds, the identification of the β integrin that functions with αV integrin is critical to the development of anti PCO therapies. Thus, this study attempted to identify the specific β subunit that heterodimerizes with αV integrin in TGFβ mediated fibrotic PCO. Both β5 and β6 integrin null lenses (LCs) show no changes in fibrotic response compared to wildtype (WT) PCS. In contrast, β8ITGcKO (β8 integrin conditional knockout) LC proliferated less and did not undergo a fibrotic response PCS. This was associated with significant attenuated canonical TGFβ and integrin signaling while the addition of active TGFβ to β8ITGcKO eyes PCS rescued both the TGFβ and integrin signaling defect and fibrotic responses. RNAseq further reveals that gremlin-1, a key profibrotic and TGFβ signaling agonist is differentially expressed between WT and β8ITGcKO LCs PCS. A therapeutic blocking antibody of αVβ8 integrin function can prevent fibrosis as it ameliorates TGFβ activation and fibrotic response by LCs PCS. We also report the effectiveness of this blocking antibody in fibrosis regression PCS. As αVβ8 integrin is a “druggable” target, the outcome of this study suggests effective therapeutics to prevent & treat fibrotic PCO.

Fibronectin has multifunctional roles in posterior capsular opacification (PCO)

Lens cells (LCs) left behind PCS robustly produce fibronectin, an extracellular matrix protein (ECM) that regulates diverse cell functions and cell signaling mechanisms including latent TGFβ complex in other fibrotic diseases. However, its roles in fibrotic PCO are not known. To determine the function of fibronectin in PCO pathogenesis, mice lacking the fibronectin gene (FN conditional knock out -FNcKO) from the lens were created. While animals from this line have normal lenses, upon lens fiber cell removal which models cataract surgery, FNcKO LCs exhibit a greatly attenuated fibrotic response from 3 days PCS onward as assessed by a reduction in surgery-induced cell proliferation, and fibrotic extracellular matrix (ECM) production and deposition. This is correlated with less upregulation of Transforming Growth Factor β (TGFβ) and integrin signaling in FNcKO LCs PCS concomitant with sustained Bone Morphogenetic Protein (BMP) signaling and elevation of the epithelial cell marker E cadherin. Although the initial fibrotic response of FNcKO LCs was qualitatively normal at 48 hours PCS as measured by the upregulation of fibrotic marker protein αSMA, RNA sequencing revealed that the fibrotic response was already quantitatively attenuated at this time, as measured by the upregulation of mRNAs encoding molecules that control, and are controlled by, TGFβ signaling, including many known markers of fibrosis. Most notably, gremlin-1, a known regulator of TGFβ superfamily signaling, was upregulated sharply in WT LCs PCS, while this response was attenuated in FNcKO LCs. As exogenous administration of either active TGFβ1 or gremlin-1 to FNcKO lens capsular bags rescued the attenuated fibrotic response of fibronectin null LCs PCS including the loss of SMAD2/3 phosphorylation, this suggests that fibronectin plays multifunctional roles in fibrotic PCO development

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