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- VEGF-B Levels in the Vitreous of Diabetic and Non-Diabetic Patients with Ocular Diseases and Its Correlation with Structural ParametersPublication . Mesquita, Joana; Sousa, João Paulo Castro De; Pereira, Sara Vaz; Neves, Arminda; Ratado, Paulo; Santos, F.M.; Passarinha, LA; Tomaz, C. T.Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples (n = 33) from diabetic (n = 25) and non-diabetic (n = 8) patients. Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL ) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (μm) was positive and moderate: rs = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm³) was positive and robust: rs = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications.
- Vascular endothelial growth factors and placenta growth factor in retinal vasculopathies: Current research and future perspectivesPublication . Mesquita, Joana; Sousa, João Paulo Castro de; Pereira, Sara Vaz; Neves, Arminda; Passarinha, L A; Tomaz, C. T.Vision loss due to disease or degeneration of the eye (retina, choroid, retinal veins, or macula) is a leading cause of blindness worldwide. In most cases, vision-threatening ocular diseases are accompanied by abnormal changes in the vasculature of the eye, especially the retina, and these conditions are collectively referred to as retinal vasculopathies. Impaired blood supply or hypoxia stimulates angiogenesis in the vascular and non-vascular sections of the eye, which results in neovascularization, leading to conditions such as diabetic retinopathy or age-related macular degeneration. Studies show that vascular endothelial growth factors: VEGF-A, VEGF-B, and placental growth factor (PlGF) are elevated in these diseases, and hence, these factors could be used as markers for disease prognosis and therapy. In this review, we discuss the function of these growth factors in normal development and disease, with focus on ocular disorders and emphasize the importance of accurately determining their levels in the vitreous and serum of patients for correct diagnosis and therapy.
- Evaluation of the growth factors VEGF-a and VEGF-B in the vitreous and serum of patients with macular and retinal vascular diseasesPublication . Mesquita, Joana; Sousa, João Paulo Castro De; Pereira, Sara Vaz; Neves, Arminda; Passarinha, L A; Tomaz, C. T.VEGF-A and VEGF-B are proangiogenic and key regulating factors for blood vessel growth. This study aims to compare VEGF-A and VEGF-B levels in the serum and vitreous of patients with neovascular pathology versus non-neovascular pathology. Our findings showed vitreous VEGF-A and VEGF-B levels increased in patients with neovascular disease, with higher levels of VEGF-A compared to VEGF-B (p ≤ .05). In the diabetic retinopathy (DR) group, higher vitreous VEGF-A or VEGF-B were found in proliferative diabetic retinopathy (PDR) than in non-PDR. The strong correlation between VEGF-A and VEGF-B demonstrates a simultaneous pathological increase of cytokines (p < .001), suggesting besides VEGF-A, VEGF-B is another contributor to ocular pathologies involving angiogenesis. There was no correlation between vitreous and serum VEGF-A or VEGF-B; however, a correlation between vitreous (VEGF-A or VEGF-B) and macular volume (p < .05) in DR patients was found. Targeting VEGF-A and VEGF-B in macular and retinal vascular diseases, involving neovascularization, may improve treatment outcomes.
- iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal DetachmentPublication . Santos, F.M.; Gaspar, Leonor Isabel Mesquita ; Ciordia, Sergio; Rocha, Ana; Sousa, João Paulo Castro De; Paradela, Alberto; Passarinha, LA; Tomaz, C. T.Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.
- Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural networkPublication . Santos, Fátima Raquel Milhano dos; Albuquerque, Tânia Gonçalves; Gaspar, Leonor Isabel Mesquita ; Dias, Joao ML; Sousa, João Paulo Castro De; Paradela, Alberto; Tomaz, C. T.; Passarinha, LADespite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.