Resilience to diabetic retinopathy.

Chronic elevation of blood glucose at first causes relatively minor changes to the neural and vascular components of the retina. As the duration of hyperglycemia persists, the nature and extent of damage increases and becomes readily detectable. While this second, overt manifestation of diabetic retinopathy (DR) has been studied extensively, what prevents maximal damage from the very start of hyperglycemia remains largely unexplored. Recent studies indicate that diabetes (DM) engages mitochondria-based defense during the retinopathy-resistant phase, and thereby enables the retina to remain healthy in the face of hyperglycemia. Such resilience is transient, and its deterioration results in progressive accumulation of retinal damage. The concepts that co-emerge with these discoveries set the stage for novel intellectual and therapeutic opportunities within the DR field. Identification of biomarkers and mediators of protection from DM-mediated damage will enable development of resilience-based therapies that will indefinitely delay the onset of DR.

Hyperglycemia Promotes Mitophagy and Thereby Mitigates Hyperglycemia-Induced Damage.

The observation that diabetic retinopathy (DR) typically takes decades to develop suggests the existence of an endogenous system that protects from diabetes-induced damage. To investigate the existance of such a system, primary human retinal endothelial cells were cultured in either normal glucose (5 mmol/L) or high glucose (30 mmol/L; HG). Prolonged exposure to HG was beneficial instead of detrimental. Although tumor necrosis factor-α-induced expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1 was unaffected after 1 day of HG, it waned as the exposure to HG was extended. Similarly, oxidative stress-induced death decreased with prolonged exposure to HG. Furthermore, mitochondrial functionality, which was compromised by 1 day of HG, was improved by 10 days of HG, and this change required increased clearance of damaged mitochondria (mitophagy). Finally, antagonizing mitochondrial dynamics compromised the cells' ability to endure HG: susceptibility to cell death increased, and basal barrier function and responsiveness to vascular endothelial growth factor deteriorated. These observations indicate the existence of an endogenous system that protects human retinal endothelial cells from the deleterious effects of HG. Hyperglycemia-induced mitochondrial adaptation is a plausible contributor to the mechanism responsible for the delayed onset of DR; loss of hyperglycemia-induced mitochondrial adaptation may set the stage for the development of DR.

On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells.

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372-384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

The Role of the Wnt Pathway in VEGF/Anti-VEGF-Dependent Control of the Endothelial Cell Barrier.

Purpose: Investigate the contribution of the Wnt pathway to vascular endothelial growth factor (VEGF)/anti-VEGF-mediated control of endothelial cell permeability. Methods: High glucose-treated primary human retinal endothelial cells (HRECs) were exposed to either VEGF, or VEGF and then anti-VEGF. Changes in gene expression were assayed by RNAseq and qRT-PCR. Permeability was monitored by electrical cell-substrate impedance sensing (ECIS). Approaches to activate the Wnt pathway included treatment with LiCl and overexpression of constitutively activated ß-catenin. ß-catenin-dependent transcriptional activity was monitored in HRECs stably expressing a TCF/LEF-driven reporter. Results: VEGF/anti-VEGF altered expression of genes encoding many members of the Wnt pathway. A subset of these genes was regulated in a way that is likely to contribute to control of the endothelial cell barrier. Namely, the VEGF-induced alteration of expression of such genes was reversed by anti-VEGF, and such adjustments occurred at times corresponding to changes in barrier function. While pharmacological and molecular approaches to activate the Wnt pathway had no effect on basal permeability, they suppressed VEGF-induced relaxation. Furthermore, anti-VEGF-mediated restoration of barrier function was unaffected by activation of the Wnt pathway. Conclusions: VEGF/anti-VEGF engages multiple members of the Wnt pathway, and activating this pathway enforces the endothelial barrier by attenuating VEGF-induced relaxation. These data suggest that FDA-approved agents such as LiCl may be an adjuvant to anti-VEGF therapy for patients afflicted with blinding conditions including diabetic retinopathy.

Analysis of Microtubule Dynamics Heterogeneity in Cell Culture.

Microtubules (MTs) are dynamic components of the cytoskeleton playing an important role in a large number of cell functions. Individual MTs in living cells undergo stochastic switching between alternate states of growth, shortening and attenuated phase, a phenomenon known as tempered dynamic instability. Dynamic instability of MTs is usually analyzed by labeling MTs with +TIPs, namely, EB proteins. Tracking of +TIP trajectories allows analyzing MT growth in cells with a different density of MTs. Numerous labs now use +TIP to track growing MTs in a variety of cell cultures. However, heterogeneity of MT dynamics is usually underestimated, and rather small sampling for the description of dynamic instability parameters is often used. The strategy described in this chapter is the method for repetitive quantitative analysis of MT growth rate within the same cell that allows minimization of the variation in MT dynamics measurement. We show that variability in MT dynamics within a cell when using repeated measurements is significantly less than between different cells in the same chamber. This approach allows better estimation of the heterogeneity of cells' responses to different treatments. To compare the effects of different MT inhibitors, the protocol using normalized values for MT dynamics and repetitive measurements for each cell is employed. This chapter provides detailed methods for analysis of MT dynamics in tissue cultures. We describe protocols for imaging MT dynamics by fluorescent microscopy, contrast enhancement technique, and MT dynamics analysis using triple color-coded display based on sequential subtraction analysis.