Effects of regular, glulisine, and aspart insulin on vascular endothelial growth factor and angiotensinogen expression in hyperglycemic retinal pigment epithelial (RPE) and human retinal endothelial cells (HRECs)

Abstract

Introduction: Diabetic retinopathy (DR) is a leading cause of vision loss and is primarily driven by chronic hyperglycemia, which induces retinal vascular damage through mechanisms involving vascular endothelial growth factor (VEGF) and the renin-angiotensin system (RAS). This study investigated the effects of hyperglycemia and different insulin formulations-regular, glulisine, and aspart-on VEGF-A and angiotensinogen (AGT) gene expression in two human retinal cell types: retinal pigment epithelial (RPE) cells and human retinal microvascular endothelial cells (HRECs).

Methods: Cells were cultured from donor tissue and exposed to physiologic and hyperglycemic glucose concentrations, with or without insulin treatment. Gene expression levels were quantified using real-time PCR.

Results: Hyperglycemia significantly upregulated VEGF-A and AGT in both RPE and HREC cells (e.g., VEGF-A in RPE: 2.62-fold, P = 0.001; AGT in RPE: 3.32-fold, P = 0.093), supporting a role for both osmotic and glucose-specific pathways. Among insulin treatments, regular insulin significantly reduced VEGF-A expression in both RPE (0.72-fold, P = 0.033) and HRECs (0.57-fold, P = 0.009). In contrast, aspart and glulisine had modest effects on VEGF-A in HRECs (0.82-fold each; P = 0.035 and P = 0.060, respectively) and no significant impact in RPE cells. Regarding AGT, aspart insulin showed the most consistent suppressive effect, reducing expression in both RPE (0.15-fold, P < 0.001) and HRECs (0.22-fold, P = 0.004). Glulisine significantly increased AGT in RPE (1.56-fold, P = 0.009) but reduced it in HRECs (0.58-fold, P = 0.074). Regular insulin showed no effect on AGT in RPE (P = 0.680) and a non-significant increase in HRECs (1.36-fold, P = 0.097).

Discussion: These findings highlight the differential biological effects of insulin analogues and suggest that aspart insulin, in particular, may offer therapeutic benefits beyond glycemic control by modulating both VEGF-A and RAS-related pathways. Tailored insulin therapies could represent innovative strategies for managing or slowing the progression of diabetic retinopathy.

Keywords: angiotensinogen; aspart; diabetic retinopathy; glulisine; human retinal endothelial cells; insulin; retinal pigment epithelium; vascular endothelial growth factor.

Figure 1

Schematic representation of the study design.

Figure 2

Expression of VEGF-A` and AGT genes under normoglycemic (physiologic; 5.5 mM, 1 g/L glucose) and hyperglycemic (25 mM, 4.5 g/L glucose) conditions in RPE and HREC cells, compared to the osmolarity control (25 mM D-mannitol; dotted line set at 1). Gene expression levels are normalized to the osmolarity control. Asterisks (*) above the error bars indicate p < 0.05 compared to the osmolarity control (one-sample t-test), while hashtags (#) above connecting lines indicate statistically significant differences between normoglycemic and hyperglycemic groups (independent t-test). RPE, retinal pigment epithelium; HREC, human retinal endothelial cells; VEGF-A, vascular endothelial growth factor-A; AGT, angiotensinogen.

Figure 3

Effect of regular, glulisine, and aspart insulin on VEGF-A expression in HREC and RPE cells under hyperglycemic conditions. Gene expression levels are normalized to the untreated hyperglycemic control (dotted line set at 1). Asterisks (*) above the error bars indicate p < 0.05 compared to the untreated hyperglycemic control (one-sample t-test). Hashtags (#) above connecting lines denote statistically significant differences between insulin-treated groups, as determined by one-way ANOVA followed by Tukey’s post hoc test. RPE, retinal pigment epithelium; HREC, human retinal endothelial cells; VEGF-A, vascular endothelial growth factor-A.

Figure 4

Effect of regular, glulisine, and aspart insulin on AGT expression in HREC and RPE cells under hyperglycemic conditions. Gene expression levels are normalized to the untreated hyperglycemic control (dotted line set at 1). Asterisks (*) above the error bars indicate p < 0.05 compared to the untreated hyperglycemic control (one-sample t-test). Hashtags (#) above connecting lines denote statistically significant differences between insulin-treated groups, as determined by one-way ANOVA followed by Tukey’s post hoc test. RPE, retinal pigment epithelium; HREC, human retinal endothelial cells; AGT, angiotensinogen.