PURPOSE. Primary open-angle glaucoma is characterized by increased resistance to aqueous humor outflow and a stiffer human trabecular meshwork (HTM). Two Yorkie homologues, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif, encoded by WWTR1 (TAZ), are mechanotransducers of the extracellular-microenvironment and coactivators of transcription. Here, we explore how substratum stiffness modulates the YAP/TAZ pathway and extracellular matrix genes in HTM cells and how this may be play a role in the onset and progression of glaucoma.METHODS. HTM cells from normal donors were cultured on hydrogels mimicking the stiffness of normal (5 kPa) and glaucomatous (75 kPa) HTM. Changes in expression of YAP/ TAZ related genes and steroid responsiveness were determined. Additionally, transglutaminase-2 expression was determined after YAP silencing.RESULTS. YAP and TAZ are both expressed in human trabecular meshwork cells. In vitro, YAP and TAZ were inversely regulated by substratum stiffness. YAP and 14-3-3r were downregulated to different extents on stiffer substrates; TAZ, tissue transglutaminase (TGM2), and soluble frizzled-related protein-1 (sFRP-1) were significantly upregulated. CTGF expression appeared to be altered differentially by both YAP and TAZ. Myocilin and angiopoietin-like 7 expression in response to dexamethasone was more pronounced on stiffer substrates. We demonstrated a direct effect by YAP on TGM2 when YAP was silenced by small interfering RNA.CONCLUSIONS. The expression of YAP/TAZ and ECM-relatedgenes is impacted on physiologically relevant substrates. YAP was upregulated in cells on softer substrates. Stiffer substrates resulted in upregulation of canonical Wnt modulators, TAZ and sFRP-1, and thus may influence the progression of glaucoma. These results demonstrate the importance of YAP/TAZ in the HTM and suggest their role in glaucoma. (Invest Ophthalmol Vis Sci.

The active engraftment of the MSCs in combination with their previously documented immunomodulatory capabilities suggests the potential for therapeutic benefit in using MSCs for the treatment of periocular and joint diseases with immune involvement.

All-trans retinoic acid (RA) is a potent inducer of regeneration. Because the liver is the principal site for storage and bioactivation of vitamin A, the current study examines the effect of RA in mouse hepatocyte proliferation and liver regeneration. Mice that received a single dose of RA (25 μg/g) by oral gavage developed hepatomegaly with increased number of Ki67-positive cells and induced expression of cell cycle genes in the liver. DNA binding data revealed that RA receptors retinoic acid receptor β (RARβ) and retinoid x receptor α (RXRα) bound to cell cycle genes Cdk1, Cdk2, Cyclin B, Cyclin E, and Cdc25a in mice with and without RA treatment. In addition, RA treatment induced novel binding of RARβ/RXRα to Cdk1, Cdk2, Cyclin D, and Cdk6 genes. All RARβ/RXRα binding sites contained AGGTCA-like motifs. RA treatment also promoted liver regeneration after partial hepatectomy (PH). RA signaling was implicated in normal liver regeneration as the mRNA levels of RARβ, Aldh1a2, Crabp1, and Crbp1 were all induced 1.5 days after PH during the active phase of hepatocyte proliferation. RA treatment prior to PH resulted in early up-regulation of RARβ, Aldh1a2, Crabp1, and Crbp1, which was accompanied by an early induction of cell cycle genes. Western blotting for RARβ, c-myc, Cyclin D, E, and A further supported the early induction of retinoid signal and cell proliferation by RA treatment. Taken together, our data suggest that RA may regulate cell cycle progression and accelerates liver regeneration. Such effect is associated with an early induction of RA signaling, which includes increased expression of the receptor, binding proteins, and processing enzyme for retinoids.

A topographically patterned substrate with stochastic surface order that closely mimics the topographic features of native basement membranes has been fabricated to investigate the influence of topographic biophysical cueing on human aortic and umbilical vein endothelial cells. The stochastic substrate was fabricated by first generating a highly porous polyelectrolyte multilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) followed by replicate production of this biomimetic topography via soft lithography. These substrates, which are easy to prepare and replicate, possess a number of prominent features associated with in vivo vascular basement membrane (interwoven ridges and grooves, bumps, and pores), which have typically been studied as singular features that frequently possess anisotropic surface order (e.g., alternating ridges and grooves). When compared to a flat surface of identical chemistry, these biomimetic topographies influenced a number of important cellular behaviors associated with the homeostasis and degradation of vascular tissues. These include modulating cell migration rate and directional persistence, proliferation rate, and gene expression associated with regulation and remodeling of vascular tissues as well as inflammation.

Mesenchymal stem cells (MSCs) represent a promising cellular therapeutic for the treatment of a variety of disorders. On transplantation, MSCs interact with diverse extracellular matrices (ECMs) that vary dramatically in topographic feature type, size and surface order. In order to investigate the impact of these topographic cues, surfaces were fabricated with either isotropically ordered holes or anisotropically ordered ridges and grooves. To simulate the biologically relevant nano through micron size scale, a series of topographically patterned substrates possessing features of differing pitch (pitch=feature width+groove width) were created. Results document that the surface order and size of substratum topographic features dramatically modulate fundamental MSC behaviors. Topographically patterned (ridge+groove) surfaces were found to significantly impact MSC alignment, elongation, and aspect ratio. Novel findings also demonstrate that submicron surfaces patterned with holes resulted in increased MSC alignment to adjacent cells as well as increased migration rates. Overall, this study demonstrates that the presentation of substratum topographic cues dramatically influence MSC behaviors in a size and shape dependent manner. The response of MSCs to substratum topographic cues was similar to other cell types that have been studied previously with regards to cell shape on ridge and groove surfaces but differed with respect to proliferation and migration. This is the first study to compare the impact of anisotropically ordered ridge and groove topographic cues to isotropically order holed topographic cues on fundamental MSC behaviors across a range of biologically relevant size scales.