A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells

Human corneal fibroblasts (HCF) and corneal stromal stem cells (CSSC) each secrete and organize a thick stroma-like extracellular matrix in response to different substrata, but neither cell type organizes matrix on tissue-culture polystyrene. This study compared cell differentiation and extracellular matrix secreted by these two cell types when they were cultured on identical substrata, polycarbonate Transwell filters. After 4 weeks in culture, both cell types upregulated expression of genes marking differentiated keratocytes (KERA, CHST6, AQP1, B3GNT7). Absolute expression levels of these genes and secretion of keratan sulfate proteoglycans were significantly greater in CSSC than HCF. Both cultures produced extensive extracellular matrix of aligned collagen fibrils types I and V, exhibiting cornea-like lamellar structure. Unlike HCF, CSSC produced little matrix in the presence of serum. Construct thickness and collagen organization was enhanced by TGF-ß3. Scanning electron microscopic examination of the polycarbonate membrane revealed shallow parallel grooves with spacing of 200–300 nm, similar to the topography of aligned nanofiber substratum which we previously showed to induce matrix organization by CSSC. These results demonstrate that both corneal fibroblasts and stromal stem cells respond to a specific pattern of topographical cues by secreting highly organized extracellular matrix typical of corneal stroma. The data also suggest that the potential for matrix secretion and organization may not be directly related to the expression of molecular markers used to identify differentiated keratocytes

PDGFRα Is a Key Regulator of T1 and T3's Differential Effect on SMA Expression in Human Corneal Fibroblasts

Purpose The goal of this study was to examine the mechanism behind the unique differential action of transforming growth factor β3 (TGF-β3) and TGF-β1 on SMA expression. It was our hypothesis that platelet-derived growth factor receptor α (PDGFRα) played a key role in determining TGF-β3's response to wounding. Methods: A stable cell line, human corneal fibroblast (HCF)-P, was created from HCFs by knocking down PDGFRα expression using a lentivirus-delivered shRNA sequence. A three-dimensional (3D) in vitro model was constructed by culturing HCF or HCF-P on poly-transwell membranes for 4 weeks in the presence and absence of 0.1 ng/mL TGF-β1 or -β3. At the end of 4 weeks, the constructs were processed for immunofluorescence and reverse transcription–quantitative polymerase chain reaction (RT-qPCR). In addition, HCF and HCF-P cell migration was evaluated. Results: In HCF, TGF-β3 treatment resulted in significantly lower α-smooth muscle actin (SMA) mRNA expression and immunolocalization when compared to TGF-β1, while in HCF-P, both TGF-β1 and -β3 treatment increased the SMA mRNA expression and immunolocalization compared to both the untreated HCF-P control and TGF-β3-treated HCF. Human corneal fibroblast-P also had a lower migration rate and construct thickness when compared to HCF. Conclusions: These results show that TGF-β3 decreases SMA in HCF, while remarkably increasing SMA in HCF-P, thus indicating that the presence or absence of PDGFRα elicits contrasting responses to the same TGF-β3 treatment. Understanding the role of PDGFRα in TGF-β3's ability to stimulate SMA may potentially help in understanding the differential functions of TGF-β1 and TGF-β3 in corneal wound healing

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

TAT-Mediated Protein Transduction into Human Corneal Epithelial Cells: p15 INK4b Inhibits Cell Proliferation and Stimulates Cell Migration

PURPOSE. The cell cycle inhibitor p15 INK4b has been localized in migrating corneal epithelial cells. In this study, TAT-fusion protein technology was used to transduce p15 INK4b into human corneal epithelial cells to examine the effect on cell proliferation and migration. METHODS. Human p15 INK4b , obtained by RT-PCR, was cloned into a TAT-HA vector, and the fusion protein was purified from bacteria transformed with the TAT-HA-p15 construct. Various dilutions of TAT-HA-p15 were applied to primary human corneal epithelial cells to test potency. In addition, the effect of exposure time was examined. Cells were labeled with bromodeoxyuridine to detect proliferation, and indirect immunofluorescence was performed. Ki67 expression was also examined. To assay cell migration, human corneal epithelial cells were plated inside a cylinder and exposed to TAT-HA-p15. The cylinder was removed, the cells were allowed to spread for 2 days, and the area of cell coverage was calculated. TAT-HA-␤-galactosidase served as the control in all experiments. Finally, the extent of retinoblastoma protein phosphorylation was assayed by Western blot in cells cultured with and without TAT-HA-p15. RESULTS. TAT-HA-p15 was successfully transduced into primary human corneal epithelial cells. TAT-HA-p15 decreased proliferation in a concentration-and time-dependent manner. The migration assay showed that TAT-HA-p15 stimulated cell migration 1.8-fold. TAT-HA-␤-galactosidase had no effect on proliferation or migration. Finally, TAT-HA-p15 decreased the level of phosphorylated retinoblastoma protein by 4.9-fold. CONCLUSIONS. Active p15 INK4b can be efficiently transduced into primary human corneal epithelial cells using TAT-fusion protein technology. p15 INK4b appears to be sufficient to inhibit corneal epithelial cell proliferation and to stimulate cell migration. (Invest Ophthalmol Vis Sci

αVβ6 Integrin Promotes Corneal Wound Healing

The lack of αVβ6 impairs basement membrane and mature hemidesmosome reformation after a superficial keratectomy wound in mouse corneas

FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring

Extracellular Vesicles Secreted by Corneal Myofibroblasts Promote Corneal Epithelial Cell Migration

Corneal epithelial wound healing is a multifaceted process that encompasses cell proliferation, migration, and communication from the corneal stroma. Upon corneal injury, bidirectional crosstalk between the epithelium and stroma via extracellular vesicles (EVs) has been reported. However, the mechanisms by which the EVs from human corneal keratocytes (HCKs), fibroblasts (HCFs), and/or myofibroblasts (HCMs) exert their effects on the corneal epithelium remain unclear. In this study, HCK-, HCF-, and HCM-EVs were isolated and characterized, and human corneal epithelial (HCE) cell migration was assessed in a scratch assay following PKH26-labeled HCK-, HCF-, or HCM-EV treatment. HCE cells proliferative and apoptotic activity following EV treatment was assessed. HCF-/HCM-EVs were enriched for CD63, CD81, ITGAV, and THBS1 compared to HCK-EV. All EVs were negative for GM130 and showed minimal differences in biophysical properties. At the proteomic level, we showed HCM-EV with a log >two-fold change in CXCL6, CXCL12, MMP1, and MMP2 expression compared to HCK-/HCF-EVs; these proteins are associated with cellular movement pathways. Upon HCM-EV treatment, HCE cell migration, velocity, and proliferation were significantly increased compared to HCK-/HCF-EVs. This study concludes that the HCM-EV protein cargo influences HCE cell migration and proliferation, and understanding these elements may provide a novel therapeutic avenue for corneal wound healing

Expression of keratocyte genes by HCF and CSSC.

<p>The absolute mRNA abundance in cells before culture (yellow) and after 4 weeks of culture without TGF-ß3 (red) or with TGF-ß3 (blue) was determined for 6 genes associated with keratocyte phenotype (ALDH3A1, AQP1, B3GNT7, CHST6, KERA, PTGDS) as described in Methods. Error bars show standard deviation of triplicate assays. Some error bars are too small to be visualized on the plot.</p

Construct thickness and cellularity.

<p>(A) Shows a graph of construct thickness at 4 weeks. TGF-ß3 treatment led to a significant increase in construct thickness for all conditions (*p<0.05). (<b>B</b>)_Graph of construct's total cells calculated from counts in optical sections of fixed cultures at 4 weeks incubation. Both cell types showed similar numbers ± TGF-ß3 when serum was present. CSSCs without serum had a significantly lower cell density (*p<0.05). Error bars = SD (n = 3).</p

Collagen Type I in constructs.

<p>Confocal micrographs demonstrating immunolocalization of type I collagen at 4 weeks in (A, B) HCF, (C, D) CSSC in serum-containing media, and (E, F) CSSC in serum-free media. Constructs treated without TGF-ß3 are on the left (A, C, E) and with TGF-ß3 are on the right (B, D, F). Bar = 50 microns.</p