Association between Insulin and Nitric Oxide in Human Retinal Microvascular Endothelial Cells in vitro

Obesity and type 2 diabetes is characterized by insulin resistance which has been reported as the major risk factors associated with the development of the endothelial dysfunction and vascular complications such as atherosclerosis. Induction of the vascular dysfunction is obviously a proved metabolic consequence of insulin resistance. Diabetes leads to altered retinal microvascular function and ultimately diabetic retinopathy. Insulin signaling may play a role in this process, and animal studies indicated a role of the insulin in the pathogenesis of retinal neovascularization through its effect on endothelial cells. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy. Diabetic retinopathy (DR) a major consequence of diabetes is considered the leading cause of vision loss and blindness worldwide among working adults. Endothelial dysfunction expediting imbalance in vascular homeostasis, is one of the primary manifestation leading to the pathogenesis of DR. NO a major vasodilator involved in the regulation of vascular homeostasis is reported to be released by insulin dependent PI3K/Akt signaling pathway. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy. Insulin stimulated NO productions are reported to be well established in cardiovascular and macrovascular endothelium and its association with PI3K/Akt pathway. Contrary to this insulin signaling in retinal endothelium have minimal reports with some studies suggesting it to be an important physiology player in hyperglycemia or insulin resistance induced DR. This prompted us to investigate the association between hyperglycemia and insulin mediated PI3K/Akt pathway eNOS directed NO production and associated multivariate effect on HRMECs survival, proliferation, angiogenesis, adhesion, apoptotic and inflammatory markers. The aim of this study is to examine the examine the effect of insulin on NO production in human retinal microvascular endothelial cells cultured in hyperglcemic conditions. In the current study in order to examine the effect of insulin on NO production, HRECs cells were cultured and grown in high glucose (30 mM) and normal (5 mM) glucose for 24 hours. Subsequently, the cells were treated with 100 nM insulin for 10 minutes, 1, 2, and 4 hours. The various parameters of PI3K/Akt signaling pathway were analyzed. IRS-1, IRS-2, PI3K, Akt, eNOS, VEFGA, NFkB were analyzed for gene expression. Adhesion molecules such as P-selectin and ICAM-1 were assessed by flow cytometry. ROS and NO production were measured by immunofluorescence and fluorometry respectively. The cell viability, cell cycle, apoptosis and total oxidative stress were evaluated by imaging flowcytometery. This study demonstrated that hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes a significant decrease in ROS and apoptosis. eNOS mediated NO production increases with hyperglycemia but remarkably decreases with insulin treatment after 1 hour, 2 hours and 4 hours. This dissimilarity of the results to previously reported studies could be due to different endothelial cell types used or varied duration of experimental hyperglycemia. The most significant reason for the variation may be due to different method of NO measurement where, most previous studies measured NO production by isolated NO meters. However, in the current study fluorometry a more sensititve method to measure oxidized product of NO, nitrite an indicator direct NO production was utilized and confirmed by the immunoflouresence technique using the dye DAF-FM Diacetate. The assessment of PI3K/Akt pathway gene expression revealed that this study demonstrates a slight increase but insignificant elevation of IRS-1 and IRS-2 genes expression in hyperglycemic condition compared to the basal control condition, while gene expression of PI3K, Akt and eNOS were significantly upregulated in the presence of high glucose. Insulin treatment caused an up regulation of IRS-1 and IRS-2 genes after 1 hour however, PI3K, Akt and eNOS were significantly reduced. The analysis of angiogenic and proapoptotic markers VEGFA and NFkB by RT-PCR showed no significant change in the expression of NFkB in hyperglycemic alone, hyperglycemic and normoglycemic cells with insulin treatment at all-time points. However, hyperglycemia significantly increased the expression of VEGF. Though compared to basal control group insulin treatment significantly increased the expression of VEGFA in both hyperglycemic and normoglycemic cells yet the expression was low compared to HG state. Consistent with the VEGFA gene expression HG significantly increased the increase the cell migration and angiogenesis while insulin treatment significantly improves barrier function. Hyperglycemia significantly increased adhesion protein P-selectin with significant reduction at 4hrs insulin treatment in the current study. This study demonstrated a significant reduction in P-selectin after insulin suggesting that insulin could participate in preventing leukocyte adhesion thereby attenuating the progression of DR. This study could not demonstrate significance change in the ICAM-1 protein. This could be due to difference in the endothelial cells used, the duration and the type of insulin treatment used. This study reported that short acting insulin commonly used in the treatment of DR could control the metabolic fluxes thereby leading to improvement in oxidative stress and apoptosis. This could prevent early changes in vasodilator, adhesion and angiogenic markers such as NO, VEGFA, P-selectin involved in the angiogenesis, inflammation and neovascularization involved in retinal vascular functioning. The study shows the potent effect of short-acting insulin treatment to counteract these biomarkers and factors involved in the pathogenesis of DR and conserving microvascular function in HRMECs exposed to hyperglycemia (30 mM) and were reported to be improved. Thus, the diabetic interventions using insulin as a key therapy with others may have the potential to be utilized as a readily available, safe and inexpensive medicine to protect against microvascular complications of DR and delay its onset. In conclusion, this study demonstrated that Hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes a significant decrease in ROS and apoptosis, eNOS mediated NO production increases with hyperglycemia but remarkably decreases with insulin treatment after 1 hour, 2 hours and 4 hours, insulin could counteract the hyperglycemic effect on AKT/pI3 kinase which mediates NO production and VEGF-A, decreased adhesion molecules p-selectin involved in barrier disorder of retinal endothelial cells. Thus it could be proposed that insulin could be considered as regulators of angiogenesis.qscienc

ASSOCIATION BETWEEN INSULIN RESISTANCE AND NITRIC OXIDE IN HUMAN RETINAL MICROVASCULAR ENDOTHELIAL CELLS IN VITRO

Diabetic retinopathy (DR) a major consequence of diabetes is considered the leading cause of vision loss and blindness worldwide among working adults. Endothelial dysfunction expediting imbalance in vascular homeostasis, is one of the primary manifestation leading to the pathogenesis of DR. NO a major vasodilator involved in the regulation of vascular homeostasis is reported to be released by insulin dependent PI3K/ Akt signaling pathway. Endothelial dysfunction impairs ocular hemodynamics by reducing the bioavailability of NO and increasing the production of reactive oxygen species (ROS) and may be responsible for the pathogenesis of vascular dysfunction in retinopathy. In the current study in order to examine the effect of insulin on NO production, HRECs cells were cultured and grown in high glucose (30mM) and normal (5mM) glucose for 24 hours. Subsequently, the cells were treated with 100nM insulin for 10 minutes, 1, 2, and 4 hours. The various parameters of PI3K/ Akt signaling pathway were analyzed. This study demonstrated that Hyperglycemia causes an increase in ROS/oxidative stress and apoptosis, while insulin promotes a significant decrease in ROS and apoptosis, eNOS mediated NO production increases with hyperglycemia but remarkably reduced with insulin treatment after 1hour, 2 hours and 4 hours. This may suggest that insulin could counteract the hyperglycemic effect on AKT/PI3 kinase which mediates NO production and VEGF-A, with decreased adhesion molecules such as p-selectin that is involved in barrier disorder of retinal endothelial cells. In summary, insulin could counteract the deleterious effects of hyperglycemia on retinal endothelial cells via various molecular approach including oxidative stress, apoptosis, NO, and adhesion molecules

Adiponectin Ameliorates Hyperglycemia-Induced Retinal Endothelial Dysfunction, Highlighting Pathways, Regulators, and Networks

Background: The pathophysiology of diabetic retinopathy (DR) is multifaced. A low level of circulating adiponectin (APN) in type 2 diabetes is associated with microvasculature complications, and its role in the evolution of DR is complex. Aim: This study is designed to explore the potential impact of APN in the pathogenesis of DR, linking the changes in cellular and biological processes with the pathways, networks, and regulators involved in its actions. Methods: Human microvascular retinal endothelial cells (HMRECs) were exposed to 30mM glucose (HG) and treated with globular adiponectin (30μg/mL) for 24 hours. The cells were evaluated for reactive oxidative stress (ROS) and apoptosis. RT-PCR profile arrays were utilized to evaluate the profile of genes involved in endothelial functions, angiogenesis, extracellular matrix, and adhesion molecules for hyperglycemic HMRECs treated with adiponectin. In addition, the barrier function, leukocyte migration, and angiogen-esis were evaluated. The differential expressed genes (DEGs) were outlined, and bioinformatic analysis was applied. Results: Adiponectin suppresses ROS production and apoptosis in HMRECs under HG conditions. Adiponectin improved migration and barrier functions in hyperglycemic cells. The bioinformatic analysis highlighted that the signaling pathways of integrin, HMGB1, and p38 AMPK, are mainly involved in the actions of APN on HMRECs. APN significantly affects molecular functions, including the adhesion of cells, chemotaxis, migration of WBCs, and angiogenesis. STAT3, NFKB, IKBKB, and mir-8 are the top upstream regulators, which affect the expressions of the genes of the data set, while TNF and TGFB1 are the top regulators. Conclusion: Adiponectin significantly counteracts hyperglycemia at various cellular and molecular levels, reducing its impact on the pathophysiological progression towards DR in vitro using HMRECs. Adiponectin ameliorates inflammatory response, oxidative stress, and endothelial barrier dysfunction using a causal network of NFBk complex, TNF, and HMGB1 and integrin pathways.We are grateful to the Biomedical Science Department, College of Health Sciences, and Biomedical Research Center, Qatar University. This study was made possible by a Qatar National Research Fund grant under its Undergraduate Research Experience Program UREP# 17-069-3-018. However, its contents are solely the authors’ responsibility and do not necessarily represent the official views of the Qatar National Research Fund. The data is available under request, and part of this work is presented in the ARVO meeting 2017, published as an abstract in Investigative Ophthalmology & Visual Science, volume 58, issue 8, pages 5225–5225

Empower Generations: Longitudinal Study for National Capacities in Life Sciences and Healthcare

Background: The decrease in the number of adolescents showing genuine interest in the fields of healthcare has been one of the recent concerns worldwide. A plethora of studies have discussed the factors that influence career choices of high school students, including science educational pedagogies, gender, environment, the student’s cognitive capabilities, and social perceptions of occupations being gender-based. As reported in 2012, a majority of the Qatari high-school students have shown a greater interest in business, technological, and administrative careers and a lower interest in healthcare. Comprehensive national and institutional strategies have since been utilized to direct the interest of Qatari generation toward healthcare careers. Objective: The primary objective of this case-control study is to assess the effect of schooling type on the enrollment in the Empower Generations (EG) career training in healthcare at the Qatar University. The secondary objectives are: (1) to describe the effect of initial career interest on the EG and healthcare majors composite’s enrollments and (2) assess the association between the history of enrollment in EG and university GPAs. Method: This is a case-control study that utilized the Qatar University’s enrollment databases for the health professions majors, that is, Health Sciences, Medicine, Pharmacy, and Dentistry. The datasets were collected from the registration records between 2013 and 2020. The statistical analysis was performed on the Statistical Package for the Social Sciences (SPSS) software version 26; the study used Chi-Square Test and Independence and logistic regression to assess the effect of schooling type and initial career interest on the enrollment in the EG training at the Qatar University. All statistics were tested for p = 0.05 and 95% CI. Results: Total QU-Health records of admissions from 2013 to 2020 involve 562 eligible students. A total of 180 students (32%) attended EG training before they were admitted to QU-Health, whereas a total of 382 (68%) were enrolled to QU-Health without attending EG training. The study revealed significant findings regarding the association between EG training and international schools (p < 0.001). Among the group who attended EG training, there were 63 students (75%) who reported that they did not have an initial career interest before they joined the EG training compared to 21 students (25%) reported that they did not have an initial career interest but enrolled immediately to healthcare majors. The findings indicate insignificant association between the history of EG training and the high school percentage p = 0.397. However, the association between a history of EG training and the university’s GPA is significant, with a p < 0.001, OR 5.016 (2.954–8.518). Conclusion: The study has shown significant association between the EG training enrollment and the type of school and the initial career interest of high school students. The EG training is perceived to direct the interest of high school students toward the careers of healthcare and is thought to enhance the performance of college students through their university’s GPAs