Novel Devices for Studying Acute and Chronic Mechanical Stress in Retinal Pigment Epithelial Cells

Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration (AMD). Overexpression of vascular endothelial growth factor (VEGF), a potent angiogenic protein, by retinal pigment epithelial (RPE) cells is a key stimulator of CNV. Mechanical stress occurs during different stages of AMD and is a possible inducer of VEGF expression in RPE cells. However, robust and realistic approaches to studying acute and chronic mechanical stress under various AMD stages do not exist.The majority of previous work has studied cyclic stretching of RPE cells grown on flexible substrates, but an ideal model must be able to mimic localized and continuous stretching of the RPE as would occur in AMD in vivo. To bridge this gap, we developed two in vitro devices to model chronic and acute mechanical stress on RPE cells during different stages of AMD. In one device, high levels of continuous mechanical stress were applied to focal regions of the RPE monolayer by stretching the underlying silicon substrate to study the role of chronic mechanical stimulation. In the second device, RPE cells were grown on porous plastic substrates and acute stress was studied by stretching small areas. Using these devices, we studied the effect of mechanical stress on VEGF expression in RPE cells.Our results suggest that mechanical stress in RPE cells inducesVEGF expression and promotes in vitro angiogenesis. These results confirm the hypothesis that mechanical stress is involved in the initiation and progression of CNV

Muscle Atrophy Marker Expression Differs between Rotary Cell Culture System and Animal Studies

Muscular atrophy, defined as the loss of muscle tissue, is a serious issue for immobilized patients on Earth and for humans during spaceflight, where microgravity prevents normal muscle loading. In vitro modeling is an important step in understanding atrophy mechanisms and testing countermeasures before animal trials. The most ideal environment for modeling must be empirically determined to best mimic known responses in vivo. To simulate microgravity conditions, murine C2C12 myoblasts were cultured in a rotary cell culture system (RCCS). Alginate encapsulation was compared against polystyrene microcarrier beads as a substrate for culturing these adherent muscle cells. Changes after culture under simulated microgravity were characterized by assessing mRNA expression of MuRF1, MAFbx, Caspase 3, Akt2, mTOR, Ankrd1, and Foxo3. Protein concentration of myosin heavy chain 4 (Myh4) was used as a differentiation marker. Cell morphology and substrate structure were evaluated with brightfield and fluorescent imaging. Differentiated C2C12 cells encapsulated in alginate had a significant increase in MuRF1 only following simulated microgravity culture and were morphologically dissimilar to normal cultured muscle tissue. On the other hand, C2C12 cells cultured on polystyrene microcarriers had significantly increased expression of MuRF1, Caspase 3, and Foxo3 and easily identifiable multinucleated myotubes. The extent of differentiation was higher in simulated microgravity and protein synthesis more active with increased Myh4, Akt2, and mTOR. The in vitro microcarrier model described herein significantly increases expression of several of the same atrophy markers as in vivo models. However, unlike animal models, MAFbx and Ankrd1 were not significantly increased and the fold change in MuRF1 and Foxo3 was lower than expected. Using a standard commercially available RCCS, the substrates and culture methods described only partially model changes in mRNAs associated with atrophy in vivo

Acute Mechanical Stress in Primary Porcine RPE Cells Induces Angiogenic Factor Expression and In Vitro Angiogenesis

Background Choroidal neovascularization (CNV) is a major cause of blindness in patients with age-related macular degeneration. CNV is characterized by new blood vessel growth and subretinal fluid accumulation, which results in mechanical pressure on retinal pigment epithelial (RPE) cells. The overexpression of RPE-derived angiogenic factors plays an important role in inducing CNV. In this work, we investigated the effect of mechanical stress on the expression of angiogenic factors in porcine RPE cells and determined the impact of conditioned medium on in-vitro angiogenesis. Results The goal of this study was to determine whether low levels of acute mechanical stress during early CNV can induce the expression of angiogenic factors in RPE cells and accelerate angiogenesis. Using a novel device, acute mechanical stress was applied to primary porcine RPE cells and the resulting changes in the expression of major angiogenic factors, VEGF, ANG2, HIF-1α, IL6, IL8 and TNF-α, were examined using immunocytochemistry, qRT-PCR, and ELISA. An in vitro tube formation assay was used to determine the effect of secreted angiogenic proteins due to mechanical stress on endothelial tube formation by human umbilical vein endothelial cells (HUVECs). Our results showed an increase in the expression of VEGF, ANG2, IL-6 and IL-8 in response to mechanical stress, resulting in increased in vitro angiogenesis. Abnormal epithelial-mesenchymal transition (EMT) in RPE cells is also associated with CNV and further retinal degeneration. Our qRT-PCR results verified an increase in the expression of EMT genes, CDH2, VIM and FN1, in RPE cells. Conclusions In conclusion, we showed that acute mechanical stress induces the expression of major angiogenic and EMT factors and promotes in vitro angiogenesis, suggesting that mechanical stress plays a role in promoting aberrant angiogenesis in AMD

Characterization of the Effects of Radiation on Skeletal and Smooth Muscle Cells

Muscular atrophy is a serious issue for extended spaceflight. Understanding and preventing the role of ionizing radiation in skeletal muscle loss would preserve the strength and endurance of astronauts and enable longer duration space travel and exploration. Irradiation was performed in the USU material physics group\u27s Space Suvivability Test Chamber. C2C12 and CRL-1999 cells were exposed to dosages ranging from 0.5 - 36.8 Gy. Cell viability and growth rate were measured immediately following irradiation

Characterizing the Effects of Radiation on Muscle Cells

One of the primary concerns for those spending time in low gravity and high radiation environments is muscle atrophy. A major cause of muscular atrophy is oxidative stress which is amplified by increased levels of ionizing radiation during spaceflight. Additionally, high levels of radiation can damage DNA, increasing the risk of cancer. Utah State University’s Space Environment Test Facility was used to irradiate C2C12 myoblasts and human vascular endothelial cells with a beta-radiation dosage mimicking that on the International Space Station and a 3-year deep space mission

Rational design of Rama-labeled nanoparticles for a dual-modaility, light scattering immunoassay on a polystyrene seubstrate

Background: Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate. Results: We have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes. Conclusion: SERS nanoparticle probes were fabricated to produce a strong light scattering signal despite substrate interference. The optical extinction and inelastic light scattering of these probes was detected by optical absorbance spectroscopy and Raman spectroscopy, respectively. This immunoassay demonstrates the feasibility of analyzing strongly enhanced Raman signals on polystyrene, which is an inexpensive yet non-ideal Raman substrate. The assay sensitivity, which is in the low pM range, suggests that these SERS probe particles could be used for Raman labeling of cell or tissue samples in a polystyrene tissue culture plate. With continued development, this approach could be used for direct labeling of multiple cell surface biomarkers on strongly interfering substrate platforms

A computational study of VEGF production by patterned retinal epithelial cell colonies as a model for neovascular macular degeneration

Background: The configuration of necrotic areas within the retinal pigmented epithelium is an important element in the progression of age-related macular degeneration (AMD). In the exudative (wet) and non-exudative (dry) forms of the disease, retinal pigment epithelial (RPE) cells respond to adjacent atrophied regions by secreting vascular endothelial growth factor (VEGF) that in turn recruits new blood vessels which lead to a further reduction in retinal function and vision. In vitro models exist for studying VEGF expression in wet AMD (Vargis et al., Biomaterials 35(13):3999–4004, 2014), but are limited in the patterns of necrotic and intact RPE epithelium they can produce and in their ability to finely resolve VEGF expression dynamics. Results: In this work, an in silico hybrid agent-based model was developed and validated using the results of this cell culture model of VEGF expression in AMD. The computational model was used to extend the cell culture investigation to explore the dynamics of VEGF expression in different sized patches of RPE cells and the role of negative feedback in VEGF expression. Results of the simulation and the cell culture studies were in excellent qualitative agreement, and close quantitative agreement. Conclusions: The model indicated that the configuration of necrotic and RPE cell-containing regions have a major impact on VEGF expression dynamics and made precise predictions of VEGF expression dynamics by groups of RPE cells of various sizes and configurations. Coupled with biological studies, this model may give insights into key molecular mechanisms of AMD progression and open routes to more effective treatments