This integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.

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.

Nesprin-3, a protein that links intermediate filaments to the nucleus, plays a role in vascular endothelial cell (EC) function. Nesprin-3 regulates EC morphology, perinuclear cytoskeletal organization, centrosome–nuclear connectivity, and flow-induced cell polarization and migration.

In vivo, vascular endothelial cells (VECs) are anchored to the underlying stroma through a specialization of the extracellular matrix, the basement membrane (BM) which provides a variety of substratum associated biophysical cues that have been shown to regulate fundamental VEC behaviors. VEC function and homeostasis are also influenced by hemodynamic cues applied to their apical surface. How the combination of these biophysical cues impacts fundamental VEC behavior remains poorly studied. In the present study, we investigated the impact of providing biophysical cues simultaneously to the basal and apical surfaces of human aortic endothelial cells (HAECs). Anisotropically ordered patterned surfaces of alternating ridges and grooves and isotropic holed surfaces of varying pitch (pitch = ridge or hole width + intervening groove or planar regions) were fabricated and seeded with HAECs. The cells were then subjected to a steady shear stress of 20 dyne/cm2 applied either parallel or perpendicular to the direction of the ridge/groove topography. HAECs subjected to flow parallel to the ridge/groove topography exhibited protagonistic effects of the two stimuli on cellular orientation and elongation. In contrast, flow perpendicular to the substrate topography resulted in largely antagonistic effects. Interestingly, the behavior depended on the shape and size of the topographic features. HAECs exhibited a response that was less influenced by the substratum and primarily driven by flow on isotropically ordered holed surfaces of identical pitch to the anistropically ordered surfaces of alternating ridges and grooves. Simultaneous presentation of biophysical cues to the basal and apical aspects of cells also influenced nuclear orientation and elongation; however, the extent of nuclear realignment was more modest in comparison to cellular realignment regardless of the surface order of topographic features. Flow-induced HAEC migration was also influenced by the ridge/groove surface topographic features with significantly altered migration direction and increased migration tortuosity when flow was oriented perpendicular to the topography; this effect was also pitch-dependent. The present findings provide valuable insight into the interaction of biologically relevant apical and basal biophysical cues in regulating cellular behavior and promise to inform improved prosthetic design.

Wnt antagonism has been linked to glaucoma and intraocular pressure regulation, as has increased stiffness of human trabecular meshwork (HTM) tissue. We have shown culturing HTM cells on substrates that mimic the elevated stiffness of glaucomatous tissue leads to elevated expression of the Wnt antagonist secreted frizzled related protein 1 (SFRP1), suggesting a linkage between SFRP1 and HTM mechanobiology. In this study, we document biomechanical consequences of Wnt antagonism on HTM cells. Cells were treated with the Wnt antagonists (SFRP1, KY02111, and LGK-974) for 8 days and allowed to recover for 4 days. After recovery, intrinsic cell stiffness and activation of the Wnt pathway via β-catenin staining and blotting were assayed. Basal cell stiffness values were 3.71±0.37, 4.33±3.07, and 3.07± kPa (median±S.D.) for cells derived from 3 donors. Cell stiffness increased after 0.25 μg/mL (4.32±5.12, 8.86±8.51, 4.84±3.15 kPa) and 0.5 μg/mL (16.75±5.59, 13.18±7.99, and 8.54±5.77 kPa) SFRP1 treatment. Stiffening was observed after 10μM KY02111 (10.72±5.63 and 6.57±5.53 kPa) as well as LGK-974 (9.60±7.41 and 11.40±9.24 kPa) treatment compared with controls (3.79±1.01 and 5.16±2.14 kPa). Additionally, Wnt inhibition resulted in decreased β-catenin staining and increased phosphorylation at threonine 41 after recovery. In conclusion, this work demonstrates a causal relationship between Wnt inhibition and cell stiffening. Additionally, these findings suggest transient Wnt inhibition resulted in durable modulation of the mechanical phenotype of HTM cells. When placed in context with previous results, these findings provide a causal link between Wnt antagonism and cell stiffness and suggest a feedback loop contributing to glaucoma progression.

The compliance of the human trabecular meshwork (HTM) has been shown to dramatically stiffen in glaucomatous patients. The purpose of this study was to determine the impact of substratum stiffness and latrunculin-B (Lat-B) on the expression and activity of the mechanotransducers, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding domain (TAZ), in primary HTM cells as the cells start to recover from Lat-B treatment. Primary human trabecular meshwork (HTM) cells were cultured on hydrogels possessing stiffness values mimicking those found in normal (5 kPa) and glaucomatous meshworks (75 kPa), or tissue culture polystyrene (TCP; >1 GPa). Cells were treated with 2.0 μM Lat-B in DMSO or DMSO alone. RT-PCR was used to determine the impact of substratum stiffness and/or Lat-B treatment on the expression of YAP, TAZ, 14-3-3σ, plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF). Immunoblotting was used to determine the expression of YAP and TAZ as well as the phosphorylation status of YAP. Immunofluorescence was used to determine YAP protein localization. YAP and TAZ mRNA expression were upregulated on the 75 kPa hydrogels in comparison to the 5 kPa hydrogels and TCP. Treatment with Lat-B resulted in a rapid and dramatic downregulation of YAP and TAZ on the 75 kPa hydrogels. On hydrogels, Lat-B treatment increased the phosphorylation of YAP at S127, while decreasing it on TCP. Similarly, Lat-B treatment resulted in markedly decreased nuclear localization of YAP on the hydrogels but elevated nuclear localization on TCP. Lat-B treatment of HTM cells on the 75 kPa hydrogels also increased 14-3-3σ mRNA, a protein important in YAP/TAZ degradation. In addition, Lat-B treatment decreased CTGF and PAI-1 mRNA on the 75 kPa hydrogels. In conclusion, substratum stiffness alters YAP/TAZ expression and YAP localization in primary HTM cells which then may modulate the expression of extracellular matrix proteins important in glaucoma. During the recovery period after Lat-B treatment, gene expression changes are more dramatic on substrates with stiffness similar to glaucomatous meshwork. Use of these hydrogels may more accurately reflect the alterations occurring in HTM cells in glaucoma after treatment with this drug.

The major histocompatibility complex (MHC), or human leukocyte antigen (HLA) gene-coding region in humans, plays a significant role in infectious disease response, autoimmunity, and cellular recognition. This super locus is essential in mate selection and kin recognition because of the organism-specific odor which can be perceived by other individuals. However, how the unique MHC genetic combination of an organism correlates with generation of the organism-specific odor is not well understood. In the present work, we have shown that human B-cells produce a set of volatile organic compounds (VOCs) that can be measured by GC-MS. More importantly, our results show that specific HLA alleles are related to production of selected VOCs, and that this leads to a cell-specific odor "fingerprint". We used a C1R HLA class I A and B locus negative cell line, along with C1R cell lines that were stably transfected with specific A and B alleles. Our work demonstrates for the first time that HLA alleles can directly influence production of specific odor compounds at the cellular level. Given that the resulting odor fingerprint depends on expression of specific HLA sequences, it may yield information on unique human scent profiles, composition of exhaled breath, as well as immune response states in future studies.

The analysis of atomic force microscopy (AFM) force data requires the selection of a contact point (CP) and is often time consuming and subjective due to influence from intermolecular forces and low signal-to-noise ratios (SNR). In this report, we present an automated algorithm for the selection of CPs in AFM force data and the evaluation of elastic moduli. We propose that the CP may be algorithmically easier to detect by identifying a linear elastic indentation region of data (high SNR) rather than the contact point itself (low SNR). Utilizing Hertzian mechanics, the data are fitted for the CP. We first detail the algorithm and then evaluate it on sample polymeric and biological materials. As a demonstration of automation, 64 x 64 force maps were analyzed to yield spatially varying topographical and mechanical information of cells. Finally, we compared manually selected CPs to automatically identified CPs and demonstrated that our automated approach is both accurate (< 10 nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show the algorithm is useful for analysis of both biomaterials and biological samples.