Joshua T. Morgan

Nesprin-3 regulates endothelial cell morphology, perinuclear cytoskeletal architecture, and flow-induced polarization

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.

Integration of basal topographic cues and apical shear stress in vascular endothelial cells

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/cm(2) 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.

Role of Substratum Stiffness in Modulating Genes Associated with Extracellular Matrix and Mechanotransducers YAP and TAZ

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 14-3-3σ 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-related-genes 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.

Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix

PURPOSE Treatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood. METHODS Primary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined. RESULTS Treatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of α-smooth muscle actin (αSMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM. DISCUSSION 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.

Substratum stiffness and latrunculin B modulate the gene expression of the mechanotransducers YAP and TAZ in human trabecular meshwork cells.

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.

Characterization of Volatile Organic Compounds in Human Leukocyte Antigen Heterologous Expression Systems: A Cell's "Chemical Odor Fingerprint"

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.

Human trabecular meshwork cells exhibit several characteristics of, but are distinct from, adipose-derived mesenchymal stem cells.

PURPOSE To support the growing promise of regenerative medicine in glaucoma, we characterized the similarities and differences between human trabecular meshwork (HTM) cells and human mesenchymal stem cells (hMSCs). METHODS HTM cells and hMSCs were phenotypically characterized by flow cytometry. Using quantitative polymerase chain reaction, the expression of myoc, angptl7, sox2, pou5f1, and notch1 was determined in both cell types with and without dexamethasone (Dex). Immunosuppressive behavior of HTM cells and hMSCs was determined using T cells activated with phytohemagglutinin. T-cell proliferation was determined using BrdU incorporation and flow cytometry. Multipotency of HTM cells and hMSCs was determined using adipogenic and osteogenic differentiation media as well as aqueous humor (AH). Alpha-smooth muscle actin (αSMA) expression was determined in HTM cells, hMSCs, and HTM tissue. RESULTS Phenotypically, HTM and hMSCs expressed CD73, CD90, CD105, and CD146 but not CD31, CD34, and CD45 and similar sox2, pou5f1, and notch1 expression. Both cell types suppressed T-cell proliferation. However, HTM cells, but not hMSCs, upregulated myoc and angptl7 in response to Dex. Additionally, HTM cells did not differentiate into adipocytes or osteocytes. Culture of hMSCs in 20%, but not 100%, AH potently induced alkaline phosphatase activity. HTM cells in culture possessed uniformly strong expression of αSMA, which contrasted with the limited expression in hMSCs and spatially discrete expression in HTM tissue. CONCLUSIONS HTM cells possess a number of important similarities with hMSCs but lack multipotency, one of the defining characteristics of stem cells. Further work is needed to explore the molecular mechanisms and functional implications underlying the phenotypic similarities.

Early responses of vascular endothelial cells to topographic cues

Vascular endothelial cells in vivo are exposed to multiple biophysical cues provided by the basement membrane, a specialized extracellular matrix through which vascular endothelial cells are attached to the underlying stroma. The importance of biophysical cues has been widely reported, but the signaling pathways that mediate cellular recognition and response to these cues remain poorly understood. Anisotropic topographically patterned substrates with nano- through microscale feature dimensions were fabricated to investigate cellular responses to topographic cues. The present study focuses on early events following exposure of human umbilical vein endothelial cells (HUVECs) to these patterned substrates. In serum-free medium and on substrates without protein coating, HUVECs oriented parallel to the long axis of underlying ridges in as little as 30 min. Immunocytochemistry showed clear differences in the localization of the focal adhesion proteins Src, p130Cas, and focal adhesion kinase (FAK) in HUVECs cultured on topographically patterned surfaces and on planar surfaces, suggesting involvement of these proteins in mediating the response to topographic features. Knockdown experiments demonstrated that FAK was not necessary for HUVEC alignment in response to topographic cues, although FAK knockdown did modulate HUVEC migration. These data identify key events early in the cellular response to biophysical stimuli.

Automated AFM force curve analysis for determining elastic modulus of biomaterials and biological samples.

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 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 × 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 (< 10nm difference between manual and automatic) and precise for non-interacting polymeric materials. Our data show that the algorithm is useful for analysis of both biomaterials and biological samples.

Robust and artifact-free mounting of tissue samples for atomic force microscopy

Immobilization of tissue-samples for atomic for microscopy (AFM) is typically done using either semi-dry tissue or by gluing the tissue sample down, both of which can introduce artifacts. Here, we describe the design of a Soft- Clamping Immobilizing Retainer of Tissue (SCIRT) for consistent and nondestructive immobilization of tissues for AFM analysis. We compare the performance of our SCIRT method with glue-immobilization for two difficult to handle tissue types: human trabecular meshwork (HTM) and rabbit cornea (RC). Our results demonstrate that the SCIRT method has several advantages, including: (i) allowing for small sample sizes, (ii) enabling continuous hydration, (iii) eliminating contact with glue or associated solvents, (iv) permitting sample recovery following measurement, and (v) ease of use. In conclusion, the SCIRT method is a simple and effective means of immobilizing soft, hydrated tissue samples consistently and without artifacts.