Browsing by Subject "Wnt signaling"
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Item Collagen Crosslinking for Keratoconus: Cellular Signaling Mechanisms(MDPI, 2023-05-16) Karamichos, Dimitrios; Nicholas, Sarah E.; Khan, Asher; Riaz, Kamran M.Collagen crosslinking (CXL) is a widely used treatment to halt the progression of keratoconus (KC). Unfortunately, a significant number of patients with progressive KC will not qualify for CXL, including those with corneas thinner than 400 microm. The present study aimed to investigate the molecular effects of CXL using in vitro models, mirroring the normal, as well as thinner corneal stroma seen in KCs. Primary human corneal stromal cells were isolated from healthy (HCFs) and keratoconus (HKCs) donors. Cells were cultured and stimulated with stable Vitamin C resulting in 3D self-assembled extracellular matrix (ECM), cell-embedded, constructs. CXL was performed on (a) thin ECM with CXL performed at week 2 and (b) normal ECM with CXL performed at week 4. Constructs without CXL served as controls. All constructs were processed for protein analysis. The results showed modulation of Wnt signaling, following CXL treatment, as measured by the protein levels of Wnt7b and Wnt10a, correlated to the expression of alpha-smooth muscle actin (SMA). Further, the expression of a recently identified KC biomarker candidate, prolactin-induced protein (PIP), was positively impacted by CXL in HKCs. CXL-driven upregulation of PGC-1 and the downregulation of SRC and Cyclin D1 in HKCs were also noted. Although the cellular/molecular impacts of CXL are largely understudied, our studies provide an approximation to the complex mechanisms of KC and CXL. Further studies are warranted to determine factors influencing CXL outcomes.Item The Role of Wnt/β-catenin Signaling in the Trabecular Meshwork Relating to Ocular Hypertensive Primary Open Angle Glaucoma(2017-05-01) Webber, Hannah C.; Mao, Weiming; Clark, Abbot F.; Pang, Iok-HouOcular hypertension is the greatest causative risk factor of primary open angle glaucoma (POAG), the most prevalent subset of age-related glaucoma. Wnt signaling antagonist sFRP1 is increased in the trabecular meshwork (TM) of patients with POAG and induces ocular hypertension in human ex vivo eyes and in mice, which is resolved upon downstream Wnt/β-catenin signaling activation. The molecular mechanisms behind this remain unknown. β-catenin plays a role as an accessory protein to classical cadherin cytosolic domains, connecting these cell-cell adhesion proteins to the actin cytoskeleton. In other cell types, Wnt/β-catenin signaling crosstalks with the TGFβ/SMAD pathway, which is overactive in the POAG TM and is implicated in ocular hypertension. Our hypothesis is that the Wnt/β-catenin signaling pathway maintains TM cell adhesion and intraocular pressure by stabilizing cadherins junctions on the TM cell membrane and by inhibiting the POAG-related TGFβ/SMAD pathway. We used primary or transformed human non-glaucomatous TM (NTM) cells for all molecular and cell-based studies. NTM cells were treated with reporter viruses to study DNA binding element activity, recombinant protein to modulate Wnt/β-catenin or TGFβ/SMAD pathways, or siRNA to knockdown pathway mediators or cadherins. After treatment, NTM nucleic acid or protein was isolated or probed for Wnt/β-catenin or TGFβ signaling markers or cadherins. Some NTM cells were also plated for Real Time Cell Analysis (RTCA) cell impedance assays. Ad5.CMV recombinant adenoviruses encoding K-cadherin and/or sFRP1 were injected into BALB/cJ mouse eyes. Conscious IOP was assessed for up to 35 days. We found that Wnt/β-catenin signaling cross-inhibits TGFβ signaling in a β-catenin and Smad4-dependent manner. This cross-inhibition resulted in a decreased K-cadherin and fibronectin expression. Wnt/β-catenin signaling also enhanced mRNA, protein, and membrane-bound levels of K-cadherin, the most highly expressed cadherin isoform in the TM. In vivo, K-cadherin reduced the ocular hypertensive effects of sFRP1. RTCA assays showed that Wnt/β-catenin signaling and K-cadherin are responsible for maintenance of TM cell adhesion. Wnt/β-catenin signaling is responsible for intraocular pressure maintenance through increased expression of K-cadherin-mediated TM cell adhesion and through inhibition of TGFβ/SMAD signaling.