9 research outputs found
Mechanisms of Abnormal Angiogenesis in Retinal Disease
Abnormal angiogenesis is often the final step towards loss of vision in many retinal diseases, including exudative age-related macular degeneration. While many risk factors have been found to be linked to elevated levels of angiogenic proteins in the eye, the exact mechanisms leading to abnormal angiogenesis remain unclear. Loss of cell-cell contact and mechanical stress are two understudied phenomena that are often associated with retinal degeneration. The hypothesis of this work is that mechanical stress and loss of cell-cell contact can promote initiation and/or progression of aberrant angiogenesis in the retina by inducing the expression of angiogenic proteins. To test this hypothesis, we used novel engineering methods, such as micropatterning, to investigate the role of intercellular junctions and mechanical stress in regulating the expression of a potent angiogenic protein, vascular endothelial growth factor (VEGF). Our results suggest that both of these phenomena can induce VEGF expression in retinal pigment epithelial (RPE) cells, a cell layer that supports the photoreceptors and maintains retinal function. In an ongoing work, we are developing a realistic model of the subretinal tissue to further study the role of cell- cell contact loss and mechanical stress in regulating angiogenic protein expression in the RPE and to determine whether these changes can lead to abnormal angiogenesis
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
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
Effect of Physical Stimuli on Angiogenic Factor Expression in Retinal Pigment Epithelial Cells
Age-related macular degeneration (AMD) is a major cause of blindness in adults. Abnormal growth of blood vessels in the eye during the course of AMD causes damage to the retina, resulting in irreversible blindness. The goal of this research was to determine whether physical pressure on retinal cells can contribute to the increased blood vessel formation. To replicate the tears in the cell layers, a micropatterning method was used as a means of detaching cells from each other. Two new devices were also developed to mimic slow and fast increases in mechanical pressure on cell layers of the eye. After detaching cells from each other and adding mechanical stress to cells, the levels of angiogenic proteins secreted by retinal cells were measured. The results showed that both cell-cell detachment and mechanical stress can increase the secretion of angiogenic proteins. After adding mechanical stress, we also added the secreted proteins to blood vessel cells and observed an increase in blood vessel formation, indicating that mechanical stress can independently induce angiogenesis. These results suggest that physical stimuli in the eye can contribute to the aberrant blood vessel formation in AMD
Protein and gene expression alteration in degenerating retinal pigment epithelial cells
Retinal pigment epithelial cells (RPEs) are highly specialized and polarized neural cells that provide support to photoreceptors. This work aims to examine growth rates, tight junction formation as well as protein and gene expression profiles of RPE cells in degeneration states. In order to mimic early to late stages of retinal degeneration, RPE cells will be contained in circular micropatterns of different sizes (100 - 500 um in diameter) created by soft lithography. In addition, RPE cells will be exposed to high glucose-induced damage to mimic retinal degeneration in diabetic retinopathy. Analysis of protein and gene expression profiles can guide us to a better understanding of molecular alterations in degenerating RPE cells which in turn can lead to the discovery of new medications specific to different stages of retinal diseases
Anaerobic E. coli fermentations as a means to better-folded recombinant proteins
Inclusion bodies are frequently formed inside the cytoplasm of Escherichia coli as a result of high-level expression of heterologous proteins. One of the most promising approaches to this problem is exploitation of folding modulators including molecular chaperones. Molecular chaperones not only prevent the formation of aggregates by facilitating the folding process, but also are capable of disassociation and refolding of inclusion bodies. The present study aimed to investigate the effect of anaerobic conditions on the expression of chaperones and subsequently on the quality of recombinant proteins expressed in E. coli. For this reason E. coli strain BL21*(DE3) was employed as the host cell for expression of a Chemotaxis protein tagged with a green fluorescent protein (CheY-GFP). Among various terminal electron acceptors used for the activation of anaerobic respiration of E. coli BL21*(DE3) fumarate and Trimethylamine N-oxide slightly improved the growth rate and biomass yield, Nitrite showed an adverse effect and nitrate and DMSO had no effect on the growth. It was concluded that the lack of a global anaerobic transcriptional regulator gene, fnr, in E. coli BL21*(DE3) was the reason for the inability of bacteria to metabolise anaerobically. To investigate the effect of oxygen limitation on protein folding, a shift from aerobic to anaerobic conditions was applied on cultures of E. coli BL21*(DE3)-pET20-CheY-GFP and the solubility of recombinant proteins was measured before and after the shift. There was no significant change in the solubility of proteins produced during aerobic conditions. In contrast, although growth was ceased upon exposure to anaerobic conditions, both fluorescence and soluble fraction of recombinant proteins were increased indicating that oxygen limitation contributes to the solubility and the refolding of insoluble recombinant proteins
Solubility Parameters of Nonelectrolyte Organic Compounds: Determination Using Quantitative Structure-Property Relationship Strategy
International audienceThe solubility parameter is considered to be a significant parameter for the chemical industry. In this study, the quantitative structure -property relationship (QSPR) method is applied to develop three models for determination of the solubility parameters of pure nonelectrolyte organic compounds at 298.15 K and atmospheric pressure. To propose comprehensive, reliable, and predictive models, about 1400 data belonging to experimental solubility parameter values of various nonelectrolyte organic compounds are studied. The genetic function approximation (GFA) mathematical approach is applied for selection of proper model parameters (molecular descriptors) and to develop a linear QSPR model. To study the nonlinear relations between the selected molecular descriptors and the solubility parameter, two approaches are pursued: the three-layer feed forward artificial neural networks (3FFANN) and the least square support vector machine (LSSVM). Furthermore, the Levenberg -Marquardt (LM) and genetic algorithm (GA) optimization methods are respectively implemented to optimize the 3FFANN and LSSVM models. Consequently, we obtain three predictive models with satisfactory results quantified by the following statistical parameters: absolute average relative deviation (AARD) of the represented/predicted properties from existing experimental values by the GFA linear equation of 4.6% and squared correlation coefficient of 0.896; AARD of the QSPR-ANN model of 3.4% and squared correlation coefficient of 0.941; and AARD of 3.1% and squared correlation coefficient of 0.947 evaluated by the QSPR-LSSVM model
Utilizing Recombinant Spider Silk Proteins to Develop a Synthetic Bruch\u27s Membrane for Modeling the Retinal Pigment Epithelium
Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch\u27s membrane. Nonporous silk membranes were prepared with comparable thicknesses (\u3c10 μm) to that of native Bruch\u27s membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC-dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like cobblestone morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch\u27s membrane and develop more biomimetic retinal models
Utilizing Recombinant Spider Silk Proteins To Develop a Synthetic Bruch’s Membrane for Modeling the Retinal Pigment Epithelium
Spider silks are intriguing biomaterials that have a high potential as innovative biomedical processes and devices. The intent of this study was to evaluate the capacity of recombinant spider silk proteins (rSSps) as a synthetic Bruch’s membrane. Nonporous silk membranes were prepared with comparable thicknesses (\u3c10 \u3eμm) to that of native Bruch’s membrane. Biomechanical characterization was performed prior to seeding cells. The ability of RPE cells (ARPE-19) to attach and grow on the membranes was then evaluated with bright-field and electron microscopy, intracellular DNA quantification, and immunocytochemical staining (ZO-1 and F-actin). Controls were cultured on permeable Transwell support membranes and characterized with the same methods. A size-dependent permeability assay, using FITC–dextran, was used to determine cell-membrane barrier function. Compared to Transwell controls, RPE cells cultured on rSSps membranes developed more native-like “cobblestone” morphologies, exhibited higher intracellular DNA content, and expressed key organizational proteins more consistently. Comparisons of the membranes to native structures revealed that the silk membranes exhibited equivalent thicknesses, biomechanical properties, and barrier functions. These findings support the use of recombinant spider silk proteins to model Bruch’s membrane and develop more biomimetic retinal models