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

Physical Disruption of Cell-Cell Contact Induces VEGF Expression in RPE Cells

PURPOSE: To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). METHODS: Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, β-catenin, and claudin-1 in RPE cultures. RESULTS: Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, β-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, β-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. CONCLUSIONS: VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in the cytoplasm of RPE cells

Synergic effect of salivary pH baselines and low pH intakes on the force relaxation of orthodontic latex elastics

Background: Latex elastics are still in common use due to their low cost and high flexibility to improve sagittal discrepancies or interdigitation of teeth. Mechanical properties of elastics are influenced by several environmental factors such as pH changes. This study evaluated similar latex elastics to define the influence of synergic effect of intermittent low pH and various baselines pH of saliva. Materials and Methods: Four groups of latex elastics (3-M Unitek, 3/16 inch) were tested (n = 15 in each group). Two groups of elastics were immersed in two tanks of artificial saliva with different pH levels of 7 and 5, and two groups were immersed in two tanks of artificial saliva with intermittent drop of pH to 4. The force was measured when the elastics were stretched to 25 mm. These measurements were taken in 0, 4, 8, 12, 24, 36, and 48 h for each group. Repeated measures analysis of variance (RMANOVA) and post-hoc Tukey's test were used to assess the findings. The level of significance was 0.05%. Results: The interaction between pH and time analyzed with RMANOVA showed no significant differences (P > 0.05) except in 36 h (P = 0.014). The Tukey's analysis showed that each comparison between any two groups did not indicate significant differences (P > 0.05) except between Groups 1 and 3 and between Groups 2 and 3 (P < 0.05). Conclusion: No significant correlation was seen between fluctuation of pH and force degradation in latex elastic band except in 36 h

Sociohistorical Narrative of Calligraphic Art in Pakistan

The development of calligraphic art in Pakistan is affected by social and historical factors. Because of the religious connotations attached to this art, its acceptance in the art circles is contentious. This qualitative paper focuses on calligraphic art done in Arabic script which developed in Pakistan after 1947. &nbsp;Exploratory research design is used for data collection.&nbsp; A narrative and descriptive approach is adopted for data presentation and analysis This paper explores calligraphic art from a historical perspective and aims to bring into light its existing status in the contemporary art circles of Pakistan.&nbsp; The results show a substantial inclination toward religious attachment in general perception and a reluctance of accepting it as a truly expressive art in artistic circles

Sociohistorical Narrative of Calligraphic Art in Pakistan

The development of calligraphic art in Pakistan is affected by social and historical factors. Because of the religious connotations attached to this art, its acceptance in the art circles is contentious. This qualitative paper focuses on calligraphic art done in Arabic script which developed in Pakistan after 1947. &nbsp;Exploratory research design is used for data collection.&nbsp; A narrative and descriptive approach is adopted for data presentation and analysis This paper explores calligraphic art from a historical perspective and aims to bring into light its existing status in the contemporary art circles of Pakistan.&nbsp; The results show a substantial inclination toward religious attachment in general perception and a reluctance of accepting it as a truly expressive art in artistic circles.</p