Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived ...mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14
CD16
CD163
CD206
immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.
Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative biointegrating materials for in situ repair and replacement of ...corneal tissue. Here, light‐curable cornea matrix (LC‐COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un‐denatured collagen and sulfated glycosaminoglycans is introduced. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross‐linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. The cross‐linked LC‐COMatrix strongly adheres to human corneas ex vivo and effectively closes full‐thickness corneal perforations with tissue loss. Likewise, in vivo, LC‐COMatrix seals large corneal perforations, replaces partial‐corneal stromal defects and biointegrates into the tissue in rabbit models. LC‐COMatrix is a natural ready‐to‐apply biointegrating adhesive that is representative of native corneal matrix with potential applications in corneal and ocular surgeries.
A light‐curable hydrogel is fabricated from decellularized porcine cornea extracellular matrix with tunable biomechanical and bioadhesive properties. The light‐curable cornea matrix (LC‐COMatrix) has appropriate viscosity for upright application on the injured cornea in an office. LC‐COMatrix is cytocompatible and effectively repairs corneal stromal and corneal perforation injuries.
Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to possess great potential in preclinical models. An efficient biomanufacturing platform is necessary ...for scale up production for clinical therapeutic applications. The aim of this study is to investigate the potential differences in neuro-regenerative properties of MSC-derived EVs generated in 2D versus 3D culture systems.
Human bone marrow MSCs (BM-MSCs) were cultured in 2D monolayer and 3D bioreactor systems. EVs were isolated using ultracentrifugation followed by size and concentration measurements utilizing dynamic light scattering (NanoSight) and by fluorescence staining (ExoView). Mouse trigeminal ganglia (TG) neurons were isolated from BALB/c mice and cultured in the presence or absence of EVs derived from 2D or 3D culture systems. Neuronal growth and morphology were monitored over 5 days followed by immunostaining for β3 tubulin. Confocal images were analyzed by Neurolucida software to obtain the density and length of the neurites.
The NanoSight tracking analysis revealed a remarkable increase (24-fold change) in the concentration of EVs obtained from the 3D versus 2D culture condition. ExoView analysis showed a significantly higher concentration of CD63, CD81, and CD9 markers in the EVs derived from 3D versus 2D conditions. Furthermore, a notable shift toward a more heterogeneous phenotype was observed in the 3D-derived EVs compared to those from 2D culture systems. EVs derived from both culture conditions remarkably induced neurite growth and elongation after 5 days in culture compared to untreated control. Neurolucida analysis of the immunostaining images (β3 tubulin) showed a significant increase in neurite length in TG neurons treated with 3D- versus 2D-derived EVs (3301.5 μm vs. 1860.5 μm, P < 0.05). Finally, Sholl analysis demonstrated a significant increase in complexity of the neuronal growth in neurons treated with 3D- versus 2D-derived EVs (P < 0.05).
This study highlights considerable differences in EVs obtained from different culture microenvironments, which could have implications for their therapeutic effects and potency. The 3D culture system seems to provide a preferred environment that modulates the paracrine function of the cells and the release of a higher number of EVs with enhanced biophysical properties and functions in the context of neurite elongation and growth.
To compare the effects of two decellularization protocols on the characteristics of fabricated COrnea Matrix (COMatrix) hydrogels. Porcine corneas were decellularized with Detergent (De) or ...Freeze-Thaw (FT)-based protocols. DNA remnant, tissue composition and α-Gal epitope content were measured. The effect of α-galactosidase on α-Gal epitope residue was assessed. Thermoresponsive and light-curable (LC) hydrogels were fabricated from decellularized corneas and characterized with turbidimetric, light-transmission and rheological experiments. The cytocompatibility and cell-mediated contraction of the fabricated COMatrices were assessed. Both protocols reduced the DNA content to < 0.1 µg/mg (native, > 0.5 µg/mg), and preserved the collagens and glycosaminoglycans. The α-Gal epitope remnant decreased by > 50% following both decellularization methods. We observed more than 90% attenuation in α-Gal epitope after treatment with α-galactosidase. The thermogelation half-time of thermoresponsive COMatrices derived from De-Based protocol (De-COMatrix) was 18 min, similar to that of FT-COMatrix (21 min). The rheological characterizations revealed significantly higher shear moduli of thermoresponsive FT-COMatrix (300.8 ± 22.5 Pa) versus De-COMatrix 178.7 ± 31.3 Pa, p < 0.01); while, this significant difference in shear moduli was preserved after fabrication of FT-LC-COMatrix and De-LC-COMatrix (18.3 ± 1.7 vs 2.8 ± 2.6 kPa, respectively, p < 0.0001). All thermoresponsive and light-curable hydrogels have similar light-transmission to human corneas. Lastly, the obtained products from both decellularization methods showed excellent in vitro cytocompatibility. We found that FT-LC-COMatrix was the only fabricated hydrogel with no significant cell-mediated contraction while seeded with corneal mesenchymal stem cells (p < 0.0001). The significant effect of decellularization protocols on biomechanical properties of hydrogels derived from porcine corneal ECM should be considered for further applications.
Arsenic Mobility and Groundwater Extraction in Bangladesh Harvey, Charles F.; Swartz, Christopher H.; Badruzzaman, A. B. M. ...
Science (American Association for the Advancement of Science),
11/2002, Volume:
298, Issue:
5598
Journal Article
Peer reviewed
High levels of arsenic in well water are causing widespread poisoning in Bangladesh. In a typical aquifer in southern Bangladesh, chemical data imply that arsenic mobilization is associated with ...recent inflow of carbon. High concentrations of radiocarbon-young methane indicate that young carbon has driven recent biogeochemical processes, and irrigation pumping is sufficient to have drawn water to the depth where dissolved arsenic is at a maximum. The results of field injection of molasses, nitrate, and low-arsenic water show that organic carbon or its degradation products may quickly mobilize arsenic, oxidants may lower arsenic concentrations, and sorption of arsenic is limited by saturation of aquifer materials.
In previous studies trace levels of perchlorate were found in lettuce (Lactuca sativa L.) irrigated with Colorado River water, which is contaminated with low levels of perchlorate from aerospace and ...defense related industries. In this paper, we report the results of a survey conducted across North America to evaluate the occurrence of perchlorate in leafy vegetables produced outside the lower Colorado River region, and evaluate the relative iodide uptake inhibition potential to perchlorate and nitrate in these leafy vegetables. Conventionally and organically produced lettuce and other leafy vegetable samples were collected from production fields and farmers' markets in the central and coastal valleys of California, New Mexico, Colorado, Michigan, Ohio, New York, Quebec, and New Jersey. Results show that 16% of the conventionally produced samples and 32% of the organically produced samples had quantifiable levels of perchlorate using ion chromatography. Estimated perchlorate exposure from organically produced leafy vegetables was approximately 2 times that of conventional produce, but generally less than 10% of the reference dose recommended by the National Academy of Sciences. Furthermore, the iodide uptake inhibition potential of perchlorate was less than 1% of that of the nitrate present. These data are consistent with those of other reported perchlorate survey work with lettuce, bottled water, breast milk, dairy milk, and human urine, and suggest a wide national presence of perchlorate.
Colonization by Staphylococcus aureus (S. aureus) has been implicated in many infectious and wound healing disorders. This study was performed to characterize the pathogenic role of S. aureus ...alpha-hemolysin (alpha-toxin) in corneal epithelial wound healing and infectious keratitis in the setting of a corneal wound. The effect of wild-type and isogenic Hla mutant (α-hemolysin gene deleted) S. aureus bacteria and conditioned media on corneal epithelial wound healing was tested in vitro using a scratch assay and in vivo using a murine epithelial debridement model. The invasiveness of wild-type and Hla mutant S. aureus was evaluated in vitro in human corneal epithelial cells and in vivo in a murine model of infectious keratitis following total epithelial debridement. S. aureus and its conditioned media significantly delayed epithelial wound closure both in vitro (P < 0.05) and in vivo (P < 0.05). The effect of S. aureus on wound healing was significantly diminished with the Hla mutant strain (P < 0.05). Likewise, compared to the wild-type strain, the Hla mutant strain demonstrated significantly reduced ability to invade corneal epithelial cells in vitro (P < 0.05) and infect murine corneas following total epithelial debridement in vivo (P < 0.05). In conclusion, S. aureus alpha-hemolysin plays a major role in the pathologic modulation of corneal epithelial wound healing and the intracellular invasion of the bacteria. Limiting colonization by S. aureus and/or blocking alpha-hemolysin may provide a therapeutic approach for corneal wound healing and infectious disorders.
•S. aureus α-hemolysin pathologically modulates corneal wound healing in vivo.•S. aureus α-hemolysin plays a major role in the intracellular invasion of S. aureus.•Murine S. aureus keratitis can be established after total epithelial debridement.
In autosomal dominant conditions with haploinsufficiency, a single functional allele cannot maintain sufficient dosage for normal function. We hypothesized that pharmacologic induction of the ...wild-type allele could lead to gene dosage compensation and mitigation of the disease manifestations. The paired box 6 (
) gene is crucial in tissue development and maintenance particularly in eye, brain, and pancreas. Aniridia is a panocular condition with impaired eye development and limited vision due to
haploinsufficiency. To test our hypothesis, we performed a chemical screen and found mitogen-activated protein kinase kinase (MEK) inhibitors to induce
expression in normal and mutant corneal cells. Treatment of newborn
-deficient mice (
) with topical or systemic MEK inhibitor PD0325901 led to increased corneal PAX6 expression, improved corneal morphology, reduced corneal opacity, and enhanced ocular function. These results suggest that induction of the wild-type allele by drug repurposing is a potential therapeutic strategy for haploinsufficiencies, which is not limited to specific mutations.
Although arsenic contaminated groundwater in Bangladesh is a serious health issue, little is known about the complex transient patterns of groundwater flow that flush solutes from aquifers and carry ...solutes into the subsurface. Hydrologic modeling results for our field site in the Munshiganj district indicate that groundwater flow is vigorous, flushing the aquifer over time-scales of decades to a century, and also transporting solute loads into the aquifer with recharge from ponds, rivers and rice fields. The combined hydrologic and biogeochemical results from our field site imply that the biogeochemistry of the aquifer system may not be in steady-state, and that the net effect of competing processes could either increase or decrease arsenic concentrations over the next decades. Modeling results suggest that irrigation has greatly changed the location, timing and chemical content of recharge to the aquifer, flushing water through the system more quickly, and also cycling large fluxes of water through rice fields during the dry season that could mobilize arsenic from oxides in near-surface sediments. Furthermore, the hydrologic model reveals that ponds, many of which have been excavated over the last 50 years, now provide much of the groundwater recharge. These ponds receive most of the waste from the villages and thus provide another potential source of organic carbon to the groundwater system.
Different approaches to delivery of mesenchymal stem/stromal cells (MSCs) for ameliorating corneal injuries have been investigated. This study was aimed to compare the efficacy of intrastromal and ...subconjunctival injection of human bone marrow-derived MSCs (hBM-MSCs) in a corneal epithelial injury model.
Twenty-four C57BL/6J mice underwent total corneal and limbal epithelial debridement. Then, the mice were divided into three different groups: (1) intrastromal hBM-MSCs injection, (2) subconjunctival hBM-MSCs injection, and (3) injection of frozen medium as a control. Mice were monitored by slit lamp and underwent anterior segment optical coherence tomography (ASOCT). Following euthanasia, the corneas were further evaluated by histology and immunostaining.
hBM-MSC injection successfully healed epithelial defects regardless of the delivery route (P < 0.001). However, intrastromal injection was superior to subconjunctival injection in reducing defect area (P = 0.001). Intrastromal injection of hBM-MSCs also significantly reduced corneal opacity and neovascularization and improved ASOCT parameters compared to subconjunctival injection or no treatment (P < 0.001, P = 0.003, and P < 0.001, respectively). Although both of the treatment groups were positive for CK12 and had reduced levels of MUC5AC compared to the control, CK12 staining was stronger in the intrastromal group compared to the subconjunctival group. Also, persistency of MSCs was confirmed by in vivo (up to 2 weeks) and in vitro assessments (up to 4 weeks).
Although the injection of hBM-MSC using both intrastromal and subconjunctival methods improve wound healing and reduce neovascularization and opacity, the intrastromal approach is superior in terms of corneal healing.
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