Understanding an Inflammatory Pathway in Diabetic Retinopathy

Diabetic Retinopathy (DR) is the leading cause of blindness in the U.S. However, not much is known of its molecular pathway and how it attributes to increases in inflammatory response in the eye. One avenue we will investigate is the transforming growth factor beta (TGFB) signaling pathway and its effect of vascular endothelial growth factor (VEGF) secretion and cell viability. VEGF is the hallmark that exacerbates DR progression in prolonged diabetes. Some major concern that have arisen are the underlying effects of oxidants and antioxidants in elevating VEGF secretion in diabetes. In attempt to learn more, we evaluated how an oxidant (acrolein) and antioxidant (hypoxia) impact 661W cone photoreceptor cells in the retina. 661W cells were cultured in DMEM, 10% FBS, 1% AB and once confluent seeded into 6 wells with 300, 000 cells per well. Cells were conditioned in 5.5 and 30mM glucose, in addition to their appropriate treatments of hypoxia induce using cobalt chloride (CoCl2) and various concentrations of acrolein including 25, 50, 100, 200uM for a 24-hr. treatment period. Following the collection of conditioned media to measure VEGF secretion and cell viability to quantify number of viable cells using the hemocytometer. Moreover, to determine the role of TGFB signaling pathway inhibition will block the molecular pathway to determine how VEGF secretion and cell viability are affected in the respective treatments listed above. Based on the data collected hypoxia has a significant impact on increasing the amount of VEGF secretion p = .002 and decrease cell viability p = .028. Additionally, acrolein played a significant role in decreasing cell viability and VEGF secretion in a dose dependent manner in 661W photoreceptor cells. Due to hypoxia and acrolein being known to affect oxidative pathways significantly, it is possible that their effects may be mediated by the TGFB pathway. Moreover, it is suggested that there is an additional increase in VEGF secretion and decrease in viable cells after inhibition of TGFB allowing us to believe that there is an additional part of the pathway that is contributing to these effects. Overall, hypoxia exerted a significant effect to reduce 661W cell viability and increase VEGF secretion and acrolein caused reduction of cell viability along with a decrease of VEGF secretion. Acrolein decreased the amount of both VEGF and cell viability in a dose dependent manner. To determine the role of TGFB signaling pathway, two inhibitors were used SMAD/SIS (1) and TGFB receptor 1 kinase (2) to inhibit the pathway from activation by inhibiting the receptor and inhibiting phosphorylation from occurring. By doing this we discovered that inhibitor 2 reduced the hypoxic induced VEGF increase in both NG and HG suggesting pathway involvement. Furthermore, we discovered that inhibitor 2, only, resulted in an increase of viable cells suggesting possible involvement as well. All in all, it seemed that the inhibitor 2 was effective in decreasing the VEGF secretion and increase viable cells to alleviate or reverse the effects seen in DR that include increased VEGF and decrease in viable cells

Understanding an inflammatory pathway in Diabetic Retinopathy

Diabetic Retinopathy (DR) is the leading cause of blindness in the U.S. However, not much is known of its molecular pathway and how it attributes to increases in inflammatory response in the eye. One avenue we will investigate is the transforming growth factor beta (TGFB) signaling pathway and its effect of vascular endothelial growth factor (VEGF) secretion and cell viability. VEGF is the hallmark that exacerbates DR progression in prolonged diabetes. Some major concern that have arisen are the underlying effects of oxidants and antioxidants in elevating VEGF secretion in diabetes. In attempt to learn more, we evaluated how an oxidant (acrolein) and antioxidant (hypoxia) impact 661W cone photoreceptor cells in the retina. 661W cells were cultured in DMEM, 10% FBS, 1% AB and once confluent seeded into 6 wells with 300, 000 cells per well. Cells were conditioned in 5.5 and 30mM glucose, in addition to their appropriate treatments of hypoxia induce using cobalt chloride (CoCl2) and various concentrations of acrolein including 25, 50, 100, 200uM for a 24-hr. treatment period. Following the collection of conditioned media to measure VEGF secretion and cell viability to quantify number of viable cells using the hemocytometer. Moreover, to determine the role of TGFB signaling pathway inhibition will block the molecular pathway to determine how VEGF secretion and cell viability are affected in the respective treatments listed above

Diabetic Retinopathy: Targeting BIGH3 to Develop Novel Molecular Therapies

Diabetic retinopathy (DR) is a complication of diabetes due to damage of blood vessels in the retina. Aside from being a major cause of blindness in the world, DR also has a significant impact on quality of life. Although there are methods to delay the progression of DR, there are no existing therapeutic regimens for early intervention. Thus, it is critical to develop cost-effective therapies towards preventing DR development

Involvement of TGFβ signaling pathway in oxidative stress and diabetic retinopathy

Diabetic Retinopathy (DR) is a leading cause of blindness in the U.S. However, not much is known of underlying molecular mechanism and how oxidative stress contributes to its development. In the present study, we investigated the involvement of TGFβ signaling pathway on the effect of oxidative stress on VEGF secretion and viability of retinal cells. VEGF is the hallmark that exacerbates DR progression in prolonged diabetes. Some major concerns that have arisen are the underlying effects of antioxidants in elevating VEGF secretion in diabetes. In this study, we evaluated how hypoxia (or low oxygen) impacts viability and VEGF secretion using 661W cone photoreceptor cells. Confluent 661W cells were grown in 5.5 mM normal or 30 mM high glucose, as well as subjected to CoCl2 to induce hypoxia. After treatment for 24 hours, conditioned media were collected for ELISA measurement to determine the amount of protein (VEGF) secretion. Viable cell numbers were also recorded. High glucose did not induce significant changes in viable cell number nor VEGF concentration in cell media. However, hypoxia condition resulted in a three-fold decrease in viable cell numbers and a three-fold increase in VEGF concentration. Furthermore, treatment with two TGFβ inhibitors: SMAD 3, SIS (or Inhibitor 1) and TGFβ receptor 1 kinase inhibitor (or Inhibitor 2) resulted in a reversal of hypoxia-induced changes. These results strongly suggest that TGFβ signaling pathway mediates hypoxia-induced retinal cell viability and VEGF secretion. Further translational research studies will provide evidence to identify appropriate and effective pharmaceutical targets in this molecular pathway to mitigate the development of DR

Investigating the Protective Role of IRBP against Oxidative Stress in Diabetic Retinopathy

Introduction: Diabetic retinopathy is an ocular condition caused by a multitude of factors as a result of elevated blood glucose. One of the primary mechanisms of damage is a result of oxidative stress. It has been found that levels of IRBP in the vitreous are decreased in the setting of diabetic retinopathy, which could be associated with damage from resulting oxidative stress. Purpose: This article will address the protective role of IRBP in diabetic retinopathy by discussing the overexpression and under expression of IRBP and its resulting effect on the retina. Increased levels of IRBP in rats with diabetic retinopathy has been shown to prevent, and even reverse, vascular permeability as well as increase neuroretinal responses. Conversely, decreased levels of IRBP are found in patients with diabetes at all stages of ocular disease, which suggests IRBP plays a role in the pathogenesis of diabetic eye disease. IRBP is produced by photoreceptors which decreases expression of IRBP in diabetic retinopathy due to damage and dysfunction. IRBP provides protection by removing retinoid byproducts, which cause oxidative damage upon accumulation. Conclusion: Judging from studies we have on human retinal cells and rats, IRBP may play a protective role against oxidative stress in the retina as well as a critical role in preservation of photoreceptors via metabolism and transport of retinoids. It is important to continue research on IRBP to further clarify this protective role in order to contribute to possible prevention and treatment of diabetic eye disease

Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Acrolein and Hypoxia Induced VEGF Secretion by 661W Cone Photoreceptors

Diabetic retinopathy (DR) is a leading cause of blindness in the United States with vascular endothelial growth factor (VEGF) being one of the main hallmarks that exacerbates this condition. In this study, we will determine the effects of acrolein and hypoxia on VEGF secretion under different glucose concentrations in mouse cone photoreceptors (661W) to study the molecular mechanisms of DR. 661W cells were cultured in P100 dishes and then seeded in 6 wells plates at 300k cells/per well. Upon confluency of 80%, cells were subjected to the appropriate treatments consisting of complete culture media with normal (NG; 5.5mM) or high glucose (HG; 30 mM). Hypoxia was induced chemically by using cobalt (ll) chloride hexahydrate solution of 300ug/ml. Acrolein concentrations consisted of low (25uM and 50uM) and high (100uM and 200uM). Elisa was used to measure the VEGF protein in conditioned media. Hypoxia had a significant impact by increasing the amount of VEGF secretion (NG=234 pg./ml, NG + hypoxia=658, HG=246, HG+ hypoxia =630; p=0.002 and 0.0275 respectively) and decrease number of viable cells (NG=2,200,000, NG + hypoxia=762,333, HG=2,230,000, HG+ hypoxia =1,040,000; p=0.003 and 0.037 respectively). Additionally, acrolein played a significant role in decreasing cell viability (p=0.035 in HG with 25uM acrolein=1,470,000, 50uM =760,200, 100uM =95,833, 200uM =76,233 and p=0.0064 in NG with 25uM acrolein=1,500,000, 50uM =1,290,000, 100uM =228,500, 200uM =58,600) and decreased VEGF secretion (p=0.05 in HG with 25uM acrolein=204 pg./ml, 50uM =112, 100uM =27.4, 200uM =18 and p=\u3c0.0001 in NG with 25uM acrolein =272, 50uM =283, 100uM =56.6, 200uM =19.4) in 661W photoreceptor cells. Hypoxia exerted significant effects to reduce 661W cell viability and increase VEGF secretion. However acrolein induced a reduction in cell viability, along with a decrease VEGF secretion. Since hypoxia (low oxygen) and acrolein (a highly reactive aldehyde) are known to affect significantly oxidative pathways, it is possible that their effects may be mediated by the TGFb pathway. Further experiments will be conducted to determine the underlying molecular mechanism