Biology subjects

Varner, V. D.

Publications and source records attributed to Varner, V. D..

3 recordsLinked to original sources

bioRxiv2024

Photo-induced changes in tissue stiffness alter epithelial budding morphogenesis in the embryonic lung

Extracellular matrix (ECM) stiffness has been shown to influence the differentiation of progenitor cells in culture, but a lack of tools to perturb the mechanical properties within intact embryonic organs has made it difficult to determine how changes in tissue stiffness influence organ patterning and morphogenesis. Photocrosslinking of the ECM has been successfully used to stiffen soft tissues, such as the cornea and skin, which are optically accessible, but this technique has not yet been applied to developing embryos. Here, we use photocrosslinking with Rose Bengal (RB) to locally and ectopically stiffen the pulmonary mesenchyme of explanted embryonic lungs cultured ex vivo. This change in mechanical properties was sufficient to suppress FGF-10-mediated budding morphogenesis along the embryonic airway, without negatively impacting patterns of cell proliferation or apoptosis. A computational model of airway branching was used to determine that FGF-10-induced buds form via a growth-induced buckling mechanism and that increased mesenchymal stiffness is sufficient to inhibit epithelial buckling. Taken together, our data demonstrate that photocrosslinking can be used to create regional differences in mechanical properties within intact embryonic organs and that these differences influence epithelial morphogenesis and patterning. Further, this photocrosslinking assay can be readily adapted to other developing tissues and model systems.

bioengineering↗

bioRxiv2024

Treatment with both TGF-β1 and PDGF-BB disrupts the stiffness-dependent myofibroblast differentiation of corneal keratocytes

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-{beta}1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-{beta}1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-{beta}1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-{beta}1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-{beta}1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-{beta}1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of -SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of -SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of -SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of SignificanceIn vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-{beta}1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated -SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB.

bioengineering↗

bioRxiv2024

Investigating transcriptional differences in mechanotransductive and ECM related genes in cultured primary corneal keratocytes, fibroblasts and myofibroblasts

PurposeAfter stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. MethodsPrimary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-{beta}1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. ResultsGenes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-{beta}1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. ConclusionsOverall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

bioengineering↗