Functional Regulation of an Oxidative Stress Mediator, Rac1, in Diabetic Retinopathy.

PURPOSE: Early activation of cytosolic NADPH oxidase-2 (Nox2) in diabetes increases retinal ROS production, damaging their mitochondria. The assembly of Nox2 holoenzyme requires activation of a small molecular weight G protein Rac1. Rac1 activation is regulated by guanine exchange factors and guanine nucleotide-dissociation inhibitors, and post-translational modifications assist in its association with exchange factors and dissociation inhibitors. The goal of this study is to investigate the mechanisms of Rac1 activation in the development of diabetic retinopathy. METHODS: The levels of the dissociation inhibitor, prenylating enzyme (farnesyltransferase, FNTA), and exchange factor Vav2 were quantified in human retinal endothelial cells, incubated in normal or high glucose for 96 h. The roles of prenylation and Vav2 in Rac1-Nox2-ROS mitochondrial damage were confirmed in FNTA-siRNA-transfected cells and using the Vav2 inhibitor EHop, respectively. Retinal histopathology and functional changes associated with diabetic retinopathy were analyzed in diabetic mice receiving EHop for 6 months. Key parameters of Rac1 activation were confirmed in the retinal microvasculature from human donors with diabetic retinopathy. RESULTS: In HRECs, glucose increased FNTA and Vav2 and decreased the dissociation inhibitor. FNTA-siRNA and EHop inhibited glucose-induced activation of Rac1-Nox2-ROS signaling. In diabetic mice, EHop ameliorated the development of retinopathy and functional/structural abnormalities and attenuated Rac1-Nox2-mitochondrial damage. Similar alterations in Rac1 regulators were observed in retinal microvasculature from human donors with diabetic retinopathy. In diabetes, Rac1 prenylation and its interactions with Vav2 contribute to Nox2-ROS-mitochondrial damage, and the pharmacological inhibitors to attenuate Rac1 interactions with its regulators could have the potential to halt/inhibit the development of diabetic retinopathy. Graphical Abstract Activation of prenylating enzyme farnesyltransferase (FNTA) in diabetes, prenylates Rac1. The binding of Rac1 with guanine nucleotide-dissociation inhibitor (GDI) is decreased, but its association with the guanine exchange factor, Vav2, is increased, resulting in Rac1 activation. Active Rac1 helps in the assembly of Nox2 holoenzyme, and Nox2 activation increases cytosolic ROS production, damaging the mitochondria. Damaged mitochondria accelerate capillary cell apoptosis, and ultimately, results in the development of diabetic retinopathy.

The Regulatory Role of Rac1, a Small Molecular Weight GTPase, in the Development of Diabetic Retinopathy.

Diabetic retinopathy, a microvascular complication of diabetes, remains the leading cause of vision loss in working age adults. Hyperglycemia is considered as the main instigator for its development, around which other molecular pathways orchestrate. Of these multiple pathways, oxidative stress induces many metabolic, functional and structural changes in the retinal cells, leading to the development of pathological features characteristic of this blinding disease. An increase in cytosolic reactive oxygen species (ROS), produced by cytosolic NADPH oxidase 2 (Nox2), is an early event in the pathogenesis of diabetic retinopathy, which leads to mitochondrial damage and retinal capillary cell apoptosis. Activation of Nox2 is mediated through an obligatory small molecular weight GTPase, Ras-related C3 botulinum toxin substrate 1 (Rac1), and subcellular localization of Rac1 and its activation are regulated by several regulators, rendering it a complex biological process. In diabetes, Rac1 is functionally activated in the retina and its vasculature, and, via Nox2-ROS, contributes to mitochondrial damage and the development of retinopathy. In addition, Rac1 is also transcriptionally activated, and epigenetic modifications play a major role in this transcriptional activation. This review focusses on the role of Rac1 and its regulation in the development and progression of diabetic retinopathy, and discusses some possible avenues for therapeutic interventions.

Inhibition of Prenylation Promotes Caspase 3 Activation, Lamin B Degradation and Loss in Metabolic Cell Viability in Pancreatic ß-Cells.

BACKGROUND/AIMS: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin B) undergo posttranslational modifications (e.g., isoprenylation) at their C-terminal cysteine residues. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Using human islets, rodent islets, and INS-1 832/13 cells, we recently reported significant metabolic defects under glucotoxic and endoplasmic reticulum (ER) stress conditions, including caspase 3 activation and lamin B degradation. The current study is aimed at further understanding the regulatory roles of protein prenylation in the induction of the aforestated metabolic defects. METHODS: Subcellular phase partitioning assay was done using Triton X-114. Cell morphology and metabolic cell viability assays were carried out using standard methodologies. RESULTS: We report that exposure of pancreatic ß-cells to Simvastatin, an inhibitor of mevalonic acid (MVA) biosynthesis, and its downstream isoprenoid derivatives, or FTI-277, an inhibitor of farnesyltransferase that mediates farnesylation of lamins, leads to activation of caspase 3 and lamin B degradation. Furthermore, Simvastatin-treatment increased activation of p38MAPK (a stress kinase) and inhibited ERK1/2 (regulator of cell proliferation). Inhibition of farnesylation also resulted in the release of degraded lamin B into the cytosolic fraction and promoted loss in metabolic cell viability. CONCLUSION: Based on these findings we conclude that protein prenylation plays key roles in islet ß-cell function. These findings affirm further support to the hypothesis that defects in prenylation pathway induce caspase-3 activation and nuclear lamin degradation in pancreatic ß-cells under the duress of metabolic stress (e.g., glucotoxicity).

Lipotoxicity augments glucotoxicity-induced mitochondrial damage in the development of diabetic retinopathy.

PURPOSE: Although hyperglycemia is the main instigator in the development of diabetic retinopathy, dyslipidemia is also considered to play an important role. In the pathogenesis of diabetic retinopathy, cytosolic NADPH oxidase 2 (Nox2) is activated before retinal mitochondria are damaged. Our aim was to investigate the effect of lipids in the development of diabetic retinopathy. METHODS: Reactive oxygen species (ROS, by 2',7'-dichlorofluorescein diacetate) and activities of Nox2 (by a lucigenin-based method) and Rac1 (by G-LISA) were quantified in retinal endothelial cells incubated with 50 µM palmitate in 5 mM glucose (lipotoxicity) or 20 mM glucose (glucolipotoxicity) for 6 to 96 hours. Mitochondrial DNA (mtDNA) damage was evaluated by extended-length PCR and its transcription by quantifying cytochrome b transcripts. RESULTS: Within 6 hours of exposure of endothelial cells to lipotoxicity, or glucotoxicity (20 mM glucose, without palmitate), significant increase in ROS, Nox2, and Rac1 was observed, which was exacerbated by glucolipotoxic insult. At 48 hours, neither lipotoxicity nor glucotoxicity had any effect on mtDNA and its transcription, but glucolipotoxicity significantly damaged mtDNA and decreased cytochrome b transcripts, and at 96 hours, glucotoxicity and glucolipotoxicity produced similar detrimental effects on mitochondrial damage. CONCLUSIONS: Although during initial exposure, lipotoxic or glucotoxic insult produces similar increase in ROS, addition of lipotoxicity in a glucotoxic environment further exacerbates ROS production, and also accelerates their damaging effects on mitochondrial homeostasis. Thus, modulation of Nox2 by pharmacological agents in prediabetic patients with dyslipidemia could retard the development of retinopathy before their hyperglycemia is observable.

Oxidative stress and epigenetic modifications in the pathogenesis of diabetic retinopathy.

Diabetic retinopathy remains the major cause of blindness among working age adults. Although a number of metabolic abnormalities have been associated with its development, due to complex nature of this multi-factorial disease, a link between any specific abnormality and diabetic retinopathy remains largely speculative. Diabetes increases oxidative stress in the retina and its capillary cells, and overwhelming evidence suggests a bidirectional relationship between oxidative stress and other major metabolic abnormalities implicated in the development of diabetic retinopathy. Due to increased production of cytosolic reactive oxygen species, mitochondrial membranes are damaged and their membrane potentials are impaired, and complex III of the electron transport system is compromised. Suboptimal enzymatic and nonenzymatic antioxidant defense system further aids in the accumulation of free radicals. As the duration of the disease progresses, mitochondrial DNA (mtDNA) is damaged and the DNA repair system is compromised, and due to impaired transcription of mtDNA-encoded proteins, the integrity of the electron transport system is encumbered. Due to decreased mtDNA biogenesis and impaired transcription, superoxide accumulation is further increased, and the vicious cycle of free radicals continues to self-propagate. Diabetic milieu also alters enzymes responsible for DNA and histone modifications, and various genes important for mitochondrial homeostasis, including mitochondrial biosynthesis, damage and antioxidant defense, undergo epigenetic modifications. Although antioxidant administration in animal models has yielded encouraging results in preventing diabetic retinopathy, controlled longitudinal human studies remain to be conducted. Furthermore, the role of epigenetic in mitochondrial homeostasis suggests that regulation of such modifications also has potential to inhibit/retard the development of diabetic retinopathy.

Metastasis suppressor NME1 regulates melanoma cell morphology, self-adhesion and motility via induction of fibronectin expression.

Expression of the metastasis suppressor NME1 in melanoma is associated with reduced cellular motility and invasion in vitro and metastasis in vivo, but the underlying molecular mechanisms are not completely understood. Herein, we report a novel mechanism through which NME1 controls melanoma cell morphology via upregulation of the extracellular matrix (ECM) protein fibronectin. Expression of NME1 strongly suppressed cell motility in melanoma cell lines 1205LU and M14. The resulting sedentary phenotype was associated with a more flattened appearance and marked increases in actin stress fibre and focal adhesion formation. NME1-induced focal adhesions were colocalized with dense deposits of fibronectin, which were absent or minimal in the corresponding NME1-deficient parental lines. NME1 was a strong inducer of fibronectin mRNA and protein expression, shown with reciprocal approaches of forced NME1 expression and shRNA-mediated knock-down. Increased synthesis and ECM deposition of fibronectin was necessary for NME1-induced cell spreading, as knock-down of fibronectin opposed the effects of NME1 on cell morphology. Fibronectin knock-down also reversed the ability of NME1 to promote aggregation when cells were plated on a non-adherent substratum. Similarly, inhibiting activation of the fibronectin receptor integrin α4ß1 with an anti-α4 antibody reversed the motility-suppressing effect of NME1. A positive correlation was observed between NME1 and fibronectin mRNA in clinical biopsies of normal skin, benign nevi and primary melanomas, but not in metastatic forms, suggesting the NME1/fibronectin axis represents a barrier to melanoma progression. In summary, these findings indicate fibronectin is an important effector of the motility-suppressing function of NME1 in melanoma cells.

Regulation of insulin secretion and reactive oxygen species production by free fatty acids in pancreatic islets.

Free fatty acids regulate insulin secretion through metabolic and intracellular signaling mechanisms such as induction of malonyl-CoA/long-chain CoA pathway, production of lipids, GPRs (G protein-coupled receptors) activation and the modulation of calcium currents. Fatty acids (FA) are also important inducers of ROS (reactive oxygen species) production in ß-cells. Production of ROS for short periods is associated with an increase in GSIS (glucose-stimulated insulin secretion), but excessive or sustained production of ROS is negatively correlated with the insulin secretory process. Several mechanisms for FA modulation of ROS production by pancreatic ß-cells have been proposed, such as the control of mitochondrial complexes and electron transport, induction of uncoupling proteins, NADPH oxidase activation, interaction with the renin-angiotensin system, and modulation of the antioxidant defense system. The major sites of superoxide production within mitochondria derive from complexes I and III. The amphiphilic nature of FA favors their incorporation into mitochondrial membranes, altering the membrane fluidity and facilitating the electron leak. The extra-mitochondrial ROS production induced by FA through the NADPH oxidase complex is also an important source of these species in ß-cells.

Small molecular weight G-protein, H-Ras, and retinal endothelial cell apoptosis in diabetes.

We have demonstrated that the expressions of small molecular weight G-protein, H-Ras, and its effector protein, Raf-1, are increased in the retina in diabetes, and the specific inhibitors of Ras function inhibit glucose-induced apoptosis of retinal capillary cells. This study is to examine the contributory roles for H-Ras in glucose-induced apoptosis of retinal endothelial cells by genetic manipulation of functionally active H-Ras levels. Bovine retinal endothelial cells were transfected with the plasmids of either wild type (WT), constitutively active (V12) or dominant-negative (N17) H-Ras. Glucose-induced increase in apoptosis, nitric oxide (NO) levels and activation of NF-kappaB and caspase-3 were determined in these genetically manipulated cells. Exposure of bovine retinal endothelial cells to 20 mM glucose significantly increased H-Ras activation as determined by Raf-1 binding assay. Overexpression of V12 in the endothelial cells further increased their glucose-induced apoptosis by 40%, NO levels by about 50%, and activated NF-kappaB and caspase-3 by about 30-40% compared to the untransfected cells incubated in 20 mM glucose. In contrast, overexpression of the inactive mutant, N17, inhibited glucose-mediated increases in apoptotic cell death, NO levels and NF-kappaB and caspase-3 activation; the values were significantly different (p < 0.02) compared to those obtained from the untransfected cells incubated under similar conditions. Our findings demonstrate that H-Ras activation is important in the activation of the specific signaling events leading to the accelerated retinal capillary cell apoptosis in hyperglycemic conditions, suggesting the possible use of H-Ras inhibitors to inhibit the pathogenesis of diabetic retinopathy.