The Anti-Inflammatory Effect of the β1-Adrenergic Receptor Antagonist Metoprolol on High Glucose Treated Human Microvascular Retinal Endothelial Cells

Hyperglycemia-induced impairment of the blood-retinal barrier represents the main pathological event in diabetic retinopathy that is elicited by a reduced cellular response to an accumulation of reactive oxygen species (ROS) and increased inflammation. The purpose of the study was to evaluate whether the selective β1-adrenoreceptor (β1-AR) antagonist metoprolol could modulate the inflammatory response to hyperglycemic conditions. For this purpose, human retinal endothelial cells (HREC) were treated with normal (5 mM) or high glucose (25 mM, HG) in the presence of metoprolol (10 μM), epinephrine (1 μM), or both compounds. Metoprolol prevented both the HG-induced reduction of cell viability (MTT assays) and the modulation of the angiogenic potential of HREC (tube formation assays) reducing the TNF-α, IL-1β, and VEGF mRNA levels (qRT-PCR). Moreover, metoprolol prevented the increase in phospho-ERK1/2, phospho-cPLA2, COX2, and protein levels (Western blot) as well as counteracting the translocation of ERK1/2 and cPLA2 (high-content screening). Metoprolol reduced ROS accumulation in HG-stimulated HREC by activating the anti-oxidative cellular response mediated by the Keap1/Nrf2/HO-1 pathway. In conclusion, metoprolol exerted a dual effect on HG-stimulated HREC, decreasing the activation of the pro-inflammatory ERK1/2/cPLA2/COX2 axis, and counteracting ROS accumulation by activating the Keap1/Nrf2/HO-1 pathway.

High Glucose-Induced Apoptosis Is Linked to Mitochondrial Connexin 43 Level in RRECs: Implications for Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most common causes of vision loss and blindness among the working-age population. High glucose (HG)-induced decrease in mitochondrial connexin 43 (mtCx43) level is known to promote mitochondrial fragmentation, cytochrome c release, and apoptosis in retinal endothelial cells associated with DR. In this study, we investigated whether counteracting HG-induced decrease in mtCx43 level would preserve mitochondrial integrity and prevent apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells grown in HG were transfected with Cx43 plasmid, or empty vector (EV), as control. Western blot (WB) analysis showed a significant decrease in mtCx43 level concomitant with increased cleaved caspase-3, Bax, cleaved PARP, and mitochondrial fragmentation in cells grown in HG condition compared to those grown in N medium. When cells grown in HG were transfected with Cx43 plasmid, mtCx43 level was significantly increased and resulted in reduced cleaved caspase-3, Bax, cleaved PARP and preservation of mitochondrial morphology with a significant decrease in the number of TUNEL-positive cells compared to those grown in HG alone. Findings from the study indicate a novel role for mtCx43 in regulating apoptosis and that maintenance of mtCx43 level could be useful in preventing HG-induced apoptosis by reducing mitochondrial fragmentation associated with retinal vascular cell loss in DR.

Dose Response of Bumetanide on Aquaporins and Angiogenesis Biomarkers in Human Retinal Endothelial Cells Exposed to Intermittent Hypoxia

Aquaporins (AQPs) are important for regulating cellular water, solute transport, and balance. Recently, AQPs have also been recognized as playing a key role in cell migration and angiogenesis. In the retina, hypoxia induces vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, resulting in retinal edema, which is facilitated by AQPs. Bumetanide is a diuretic agent and AQP 1–4 blocker. We tested the hypothesis that bumetanide suppression of AQPs ameliorates intermittent hypoxia (IH)-induced angiogenesis and oxidative stress in human microvascular retinal endothelial cells (HMRECs). HMRECs were treated with a low-dose (0.05 µg/mL) or high-dose (0.2 µg/mL) of bumetanide and were exposed to normoxia (Nx), hyperoxia (50% O2), or IH (50% O2 with brief hypoxia 5% O2) for 24, 48, and 72 h. Angiogenesis and oxidative stress biomarkers were determined in the culture media, and the cells were assessed for tube formation capacity and AQP-1 and -4 expression. Both doses of bumetanide significantly decreased oxidative stress and angiogenesis biomarkers. This response was reflected by reductions in tube formation capacity and AQP expression. These findings confirm the role of AQPs in retinal angiogenesis. Therapeutic targeting of AQPs with bumetanide may be advantageous for IH-induced aberrant retinal development.

Interleukin-6 Trans-Signaling Mediated Regulation of Paracellular Permeability in Human Retinal Endothelial Cells

Long-term hyperglycemia-mediated oxidative stress and inflammation lead to the blood-retinal barrier (BRB) dysfunction and increased vascular permeability associated with diabetic retinopathy (DR). Interleukin-6 (IL-6) is one of the primary mediators of retinal vascular inflammation. IL-6 signaling through its membrane-bound IL-6 receptor is known as classical signaling, and through a soluble IL-6 receptor (sIL-6R) is known as trans-signaling. Increasing evidence suggests that classical signaling is primarily anti-inflammatory, whereas trans-signaling induces the pro-inflammatory effects of IL-6. The purpose of this study was to compare the effects of these two pathways on paracellular permeability and expression of genes involved in inter-endothelial junctions in human retinal endothelial cells (HRECs). IL-6 trans-signaling activation caused significant disruption to paracellular integrity, with increased paracellular permeability, and was associated with significant changes in gene expression related to adherens, tight, and gap junctions. IL-6 classical signaling did not alter paracellular resistance in HRECs and had no distinct effects on gene expression. In conclusion, IL-6 trans-signaling, but not classical signaling, is a major mediator of the increased paracellular permeability characteristic of inner BRB breakdown in diabetic retinopathy. This study also identified potential inter-endothelial junction genes involved in the IL-6 trans-signaling mediated regulation of paracellular permeability in HRECs.

Celastrol inhibits the proliferation and angiogenesis of high glucose-induced human retinal endothelial cells

Background Diabetic retinopathy (DR) is one of the most common microvascular complications of diabetes. Celastrol plays a certain role in the improvement of various diabetes complications. Therefore, this study aimed to explore whether celastrol inhibited the proliferation and angiogenesis of high glucose (HG)-induced human retinal endothelial cells (hRECs) by down-regulating the HIF1/VEGF signaling pathway. Methods The viability and proliferation of hRECs treated with glucose, celastrol or dimethyloxallyl glycine (DMOG) were analyzed by MTT assay. The invasion and tube formation ability of hRECs treated with glucose, celastrol or DMOG were in turn detected by transwell assay and tube formation assay. The expression of HIF1α and VEGF in hRECs after indicated treatment was analyzed by Western blot analysis and RT-qPCR analysis and ICAM-1 expression in hRECs after indicated treatment was detected by immunofluorescence assay Results HG induction promoted the proliferation, invasion and tube formation ability and increased the expression of HIF-1α and VEGF of hRECs, which were gradually suppressed by celastrol changing from 0.5 to 2.0 μM. DMOG was regarded as a HIF1α agonist, which attenuated the effect of celastrol on HG-induced hRECs. Conclusion Celastrol inhibited the proliferation and angiogenesis of HG-induced hRECs by down-regulating the HIF1α/VEGF signaling pathway.