RhoA/ROCK signaling regulates TGFß-induced epithelial-mesenchymal transition of lens epithelial cells through MRTF-A.

Transforming growth factor (TGF)-ß-induced epithelial-mesenchymal transition (EMT) leads to the formation of ocular fibrotic pathologies, such as anterior subcapsular cataract and posterior capsule opacification. Remodeling of the actin cytoskeleton, mediated by the Rho family of GTPases, plays a key role in EMT, however, how actin dynamics affect downstream markers of EMT has not been fully determined. Our previous work suggests that myocardin related transcription factor A (MRTF-A), an actin-binding protein, might be an important mediator of TGFß-induced EMT in lens epithelial cells. The aim of the current study was to determine the requirement of RhoA/ROCK signaling in mediating TGFß-induced nuclear accumulation of MRTF-A, and ultimate expression of α-smooth muscle actin (αSMA), a marker of a contractile, myofibroblast phenotype. Using rat lens epithelial explants, we demonstrate that ROCK inhibition using Y-27632 prevents TGFß-induced nuclear accumulation of MRTF-A, E-cadherin/ß-catenin complex disassembly, and αSMA expression. Using a novel inhibitor specifically targeting MRTF-A signaling, CCG-203971, we further demonstrate the requirement of MRTF-A nuclear localization and activity in the induction of αSMA expression. Overall, our findings suggest that TGFß-induced cytoskeletal reorganization through RhoA/ROCK/MRTF-A signaling is critical to EMT of lens epithelial cells.

Matrix metalloproteinase-9-null mice are resistant to TGF-ß-induced anterior subcapsular cataract formation.

Epithelial-mesenchymal transition (EMT) is associated with fibrotic diseases in the lens, such as anterior subcapsular cataract (ASC) formation. Often mediated by transforming growth factor (TGF)-ß, EMT in the lens involves the transformation of lens epithelial cells into a multilayering of myofibroblasts, which manifest as plaques beneath the lens capsule. TGF-ß-induced EMT and ASC have been associated with the up-regulation of two matrix metalloproteinases (MMPs): MMP-2 and MMP-9. The current study used MMP-2 and MMP-9 knockout (KO) mice to further determine their unique roles in TGF-ß-induced ASC formation. Adenoviral injection of active TGF-ß1 into the anterior chamber of all wild-type and MMP-2 KO mice led to the formation of distinct ASC plaques that were positive for α-smooth muscle actin, a marker of EMT. In contrast, only a small proportion of the MMP-9 KO eyes injected with adenovirus-expressing TGF-ß1 exhibited ASC plaques. Isolated lens epithelial explants from wild-type and MMP-2 KO mice that were treated with TGF-ß exhibited features indicative of EMT, whereas those from MMP-9 KO mice did not acquire a mesenchymal phenotype. MMP-9 KO mice were further bred onto a TGF-ß1 transgenic mouse line that exhibits severe ASC formation, but shows a resistance to ASC formation in the absence of MMP-9. These findings suggest that MMP-9 expression is more critical than MMP-2 in mediating TGF-ß-induced ASC formation.

Differential Impact of Biomechanical Constraints on Control Signal Dimensionality for Gravity Support Versus Propulsion.

Neural control of movement has to overcome the problem of redundancy in the multidimensional musculoskeletal system. The problem can be solved by reducing the dimensionality of the control space of motor commands, i.e., through muscle synergies or motor primitives. Evidence for this solution exists, multiple studies have obtained muscle synergies using decomposition methods. These synergies vary across different workspaces and are present in both dominant and non-dominant limbs. Here we explore the dimensionality of control space by examining muscle activity patterns across reaching movements in different directions starting from different postures performed bilaterally by healthy individuals. We further explore the effect of biomechanical constraints on the dimensionality of control space. We are building on top of prior work showing that muscle activity profiles can be explained by applied moments about the limb joints that reflect the biomechanical constraints. These muscle torques derived from motion capture represent the combined actions of muscle contractions that are under the control of the nervous system. Here we test the generalizability of the relationship between muscle torques and muscle activity profiles across different starting positions and between limbs. We also test a hypothesis that the dimensionality of control space is shaped by biomechanical constraints. We used principal component analysis to evaluate the contribution of individual muscles to producing muscle torques across different workspaces and in both dominant and non-dominant limbs. Results generalize and support the hypothesis. We show that the muscle torques that support the limb against gravity are produced by more consistent combinations of muscle co-contraction than those that produce propulsion. This effect was the strongest in the non-dominant arm moving in the lateral workspace on one side of the body.

Setup for the Quantitative Assessment of Motion and Muscle Activity During a Virtual Modified Box and Block Test.

The ability to move allows us to interact with the world. When this ability is impaired, it can significantly reduce one's quality of life and independence and may lead to complications. The importance of remote patient evaluation and rehabilitation has recently grown due to limited access to in-person services. For example, the COVID-19 pandemic unexpectedly resulted in strict regulations, reducing access to non-emergent healthcare services. Additionally, remote care offers an opportunity to address healthcare disparities in rural, underserved, and low-income areas where access to services remains limited. Improving accessibility through remote care options would limit the number of hospital or specialist visits and render routine care more affordable. Finally, the use of readily available commercial consumer electronics for at-home care can enhance patient outcomes due to improved quantitative observation of symptoms, treatment efficacy, and therapy dosage. While remote care is a promising means to address these issues, there is a crucial need to quantitatively characterize motor impairment for such applications. The following protocol seeks to address this knowledge gap to enable clinicians and researchers to obtain high-resolution data on complex movement and underlying muscle activity. The ultimate goal is to develop a protocol for remote administration of functional clinical tests. Here, participants were instructed to perform a medically-inspired Box and Block task (BBT), which is frequently used to assess hand function. This task requires subjects to transport standardized cubes between two compartments separated by a barrier. We implemented a modified BBT in virtual reality to demonstrate the potential of developing remote assessment protocols. Muscle activation was captured for each subject using surface electromyography. This protocol allowed for the acquisition of high-quality data to better characterize movement impairment in a detailed and quantitative manner. Ultimately, these data have the potential to be used to develop protocols for virtual rehabilitation and remote patient monitoring.

Nuclear translocation of myocardin-related transcription factor-A during transforming growth factor beta-induced epithelial to mesenchymal transition of lens epithelial cells.

PURPOSE: Transforming growth factor beta (TGFß) is a known inducer of epithelial to mesenchymal transition (EMT), and studies in other systems have shown that nuclear localization of the myocardin-related transcription factor (MRTF) is downstream of TGFß. In the following study, we investigated whether nuclear translocation of MRTF-A or MRTF-B is involved in TGFß-induced EMT of lens epithelial cells (LECs). We further investigated the relationship between matrix metalloproteinase-2 and -9 (MMP-2/9) and MRTF in the EMT of LECs. METHODS: Rat lens explant cultures were used as the model system. Explants were treated with TGFß, an MMP-2/9 inhibitor, or actin binding drugs and immunostained for alpha smooth muscle actin (αSMA), MRTF-A, and MRTF-B. Cytoplasmic and nuclear intensities of cells were measured using ImageJ. Production of αSMA was measured using western blot analysis and ImageJ. RESULTS: Untreated explant cells exhibited little αSMA expression, and MRTF-A and B were found to reside primarily in the cytosol. However, when stimulated with TGFß, a significantly greater number of cells exhibited nuclear expression of MRTF-A, accompanied by an increase in αSMA expression. However, MRTF-B remained in the cytoplasm following TGFß treatment. Cotreatment with an MMP-2/9 inhibitor and TGFß resulted in reduced MRTF-A nuclear localization and αSMA expression compared to cells treated with TGFß alone. CONCLUSIONS: Our results are the first to demonstrate the expression of MRTF-A in LECs and that its nuclear translocation can be stimulated by TGFß. Our data further suggest that MMP-2 and -9 are involved in the translocation of MRTF-A in LECs during TGFß-induced EMT.

Overlapping expression patterns and redundant roles for AP-2 transcription factors in the developing mammalian retina.

BACKGROUND: We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2ß and AP-2γ, showed expression patterns similar to AP-2α. RESULTS: Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2ß are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/ß-immunopositive population. To address possible redundant roles for AP-2α and AP-2ß during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone. CONCLUSIONS: These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.

Machine learning for spatial stratification of progressive cardiovascular dysfunction in a murine model of type 2 diabetes mellitus.

Speckle tracking echocardiography (STE) has been utilized to evaluate independent spatial alterations in the diabetic heart, but the progressive manifestation of regional and segmental cardiac dysfunction in the type 2 diabetic (T2DM) heart remains understudied. Therefore, the objective of this study was to elucidate if machine learning could be utilized to reliably describe patterns of the progressive regional and segmental dysfunction that are associated with the development of cardiac contractile dysfunction in the T2DM heart. Non-invasive conventional echocardiography and STE datasets were utilized to segregate mice into two pre-determined groups, wild-type and Db/Db, at 5, 12, 20, and 25 weeks. A support vector machine model, which classifies data using a single line, or hyperplane, that best separates each class, and a ReliefF algorithm, which ranks features by how well each feature lends to the classification of data, were used to identify and rank cardiac regions, segments, and features by their ability to identify cardiac dysfunction. STE features more accurately segregated animals as diabetic or non-diabetic when compared with conventional echocardiography, and the ReliefF algorithm efficiently ranked STE features by their ability to identify cardiac dysfunction. The Septal region, and the AntSeptum segment, best identified cardiac dysfunction at 5, 20, and 25 weeks, with the AntSeptum also containing the greatest number of features which differed between diabetic and non-diabetic mice. Cardiac dysfunction manifests in a spatial and temporal fashion, and is defined by patterns of regional and segmental dysfunction in the T2DM heart which are identifiable using machine learning methodologies. Further, machine learning identified the Septal region and AntSeptum segment as locales of interest for therapeutic interventions aimed at ameliorating cardiac dysfunction in T2DM, suggesting that machine learning may provide a more thorough approach to managing contractile data with the intention of identifying experimental and therapeutic targets.

Dual function of TGFß in lens epithelial cell fate: implications for secondary cataract.

The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFß has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified. We show here for the first time that TGFß can induce purified primary lens epithelial cells within the same culture to undergo differentiation into either lens fiber cells or myofibroblasts. Marker analysis confirmed that the two cell phenotypes were mutually exclusive. Blocking the p38 kinase pathway, either with direct inhibitors of the p38 MAP kinase or a small-molecule therapeutic that also inhibits the activation of p38, prevented TGFß from inducing epithelial-myofibroblast transition and cell migration but did not prevent fiber cell differentiation. Rapamycin had the converse effect, linking MTOR signaling to induction of fiber cell differentiation by TGFß. In addition to providing novel potential therapeutic strategies for PCO, our findings extend the so-called TGFß paradox, in which TGFß can induce two disparate cell fates, to a new epithelial disease state.

ß-Catenin/CBP-Dependent Signaling Regulates TGF-ß-Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells.

PURPOSE: Transforming growth factor-ß-induced epithelial-mesenchymal transition (EMT) is one of the main causes of posterior capsular opacification (PCO) or secondary cataract; however, the signaling events involved in TGF-ß-induced PCO have not been fully characterized. Here, we focus on examining the role of ß-catenin/cyclic AMP response element-binding protein (CREB)-binding protein (CBP) and ß-catenin/T-cell factor (TCF)-dependent signaling in regulating cytoskeletal dynamics during TGF-ß-induced EMT in lens epithelial explants. METHODS: Rat lens epithelial explants were cultured in medium M199 in the absence of serum. Explants were treated with TGF-ß2 in the presence or absence of the ß-catenin/CBP interaction inhibitor, ICG-001, or the ß-catenin/TCF interaction inhibitor, PNU-74654. Western blot and immunofluorescence experiments were carried out and analyzed. RESULTS: An increase in the expression of fascin, an actin-bundling protein, was observed in the lens explants upon stimulation with TGF-ß, and colocalized with F-actin filaments. Inhibition of ß-catenin/CBP interactions, but not ß-catenin/TCF interactions, led to a decrease in TGF-ß-induced fascin and stress fiber formation, as well as a decrease in the expression of known markers of EMT, α-smooth muscle actin (α-SMA) and matrix metalloproteinase 9 (MMP9). In addition, inhibition of ß-catenin/CBP-dependent signaling also prevented TGF-ß-induced downregulation of epithelial cadherin (E-cadherin) in lens explants. CONCLUSIONS: We show that ß-catenin/CBP-dependent signaling regulates fascin, MMP9, and α-SMA expression during TGF-ß-induced EMT. We demonstrate that ß-catenin/CBP-dependent signaling is crucial for TGF-ß-induced EMT in the lens.