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.

Bumetanide Suppression of Angiogenesis in a Rat Model of Oxygen-Induced Retinopathy.

Aquaporins (AQPs) are involved in hypoxia-induced angiogenesis and retinal damage. Bumetanide is a diuretic agent, Na+/K+/Cl- cotransporter (NKCC1), and AQP 1-4 inhibitor. We tested the hypothesis that early postnatal treatment with bumetanide suppresses biomarkers of angiogenesis and decreases severe retinopathy oxygen-induced retinopathy (OIR). Neonatal rats were exposed at birth (P0) to either (1) room air (RA); (2) hyperoxia (50% O2); or (3) intermittent hypoxia (IH) consisting of 50% O2 with brief, clustered episodes of 12% O2 from P0 to postnatal day 14 (P14), during which they were treated intraperitoneally (IP) with bumetanide (0.1 mg/kg/day) or an equivalent volume of saline, on P0-P2. Pups were examined at P14 or allowed to recover in RA from P14-P21. Retinal angiogenesis, morphometry, pathology, AQPs, and angiogenesis biomarkers were determined at P14 and P21. Bumetanide reduced vascular abnormalities associated with severe OIR. This was associated with reductions in AQP-4 and VEGF. Bumetanide suppressed sVEGFR-1 in the serum and vitreous fluid, but levels were increased in the ocular tissues during recovery. Similar responses were noted for IGF-I. In this model, early systemic bumetanide administration reduces severe OIR, the benefits of which appear to be mediated via suppression of AQP-4 and VEGF. Further studies are needed to determine whether bumetanide at the right doses may be considered a potential pharmacologic agent to treat retinal neovascularization.

Impact of Chronic Neonatal Intermittent Hypoxia on Severity of Retinal Damage in a Rat Model of Oxygen-Induced Retinopathy.

Neonatal intermittent hypoxia (IH) followed by re-oxygenation in normoxia or supplemental oxygen (IHR) increases the risk for severe retinopathy of prematurity (ROP). The exact timing for the onset of retinal damage which may guide strategic interventions during retinal development, is unknown. We tested the hypothesis that chronic exposure of the immature retina to neonatal IH induces early manifestations of retinal damage that can be utilized as key time points for strategic pharmacologic intervention. Newborn rats were exposed to IH within 2 hours of birth (P0) until P14, or allowed to recover in room air (RA) from P14 to P21 (IHR). Retinal integrity and angiogenesis biomarkers were progressively assessed before (P0), during IH, and post IH (recovery in RA), or IHR, and compared to normoxic age-matched controls. Retinal damage occurred as early as day 3 of neonatal IH, consistent with vascular abnormalities and disturbances in the astrocytic template. These abnormalities worsened during IHR. Pharmacologic and non-pharmacologic interventions to identify, prevent, or minimize neonatal IH should be implemented shortly after birth in high risk preterm newborns. This strategy may lead to a reduction in the outcome of severe ROP requiring later invasive treatments.

Associated inflammation or increased flow-mediated shear stress, but not pressure alone, disrupts endothelial caveolin-1 in infants with pulmonary hypertension.

Endothelial caveolin-1 loss is an important feature of pulmonary hypertension (PH); the rescue of caveolin-1 abrogates experimental PH. Recent studies in human PH suggest that the endothelial caveolin-1 loss is followed by an enhanced expression of caveolin-1 in smooth muscle cells (SMC) with subsequent neointima formation. In order to evaluate caveolin-1 expression in infants with PH, we examined the available clinical histories, hemodynamic data, and the expression of caveolin-1, PECAM-1, vWF, and smooth muscle α-actin in the lung biopsy/autopsy specimens obtained from infants with congenital heart disease (CHD, n = 8) and lung disease (n = 9). In CHD group, PH associated with increased pulmonary blood flow exhibited loss of endothelial caveolin-1 and PECAM-1 in pulmonary arteries; additional vWF loss was associated with enhanced expression of caveolin-1 in SMC. In the absence of PH, increased or decreased pulmonary blood flow did not disrupt endothelial caveolin-1, PECAM-1, or vWF; nor was there any enhanced expression of caveolin-1 in SMC. In Lung Disease + PH group, caveolin-1, PECAM-1, and vWF were well preserved in seven infants, and importantly, SMC in these arteries did not exhibit enhanced caveolin-1 expression. Two infants with associated inflammatory disease exhibited loss of endothelial caveolin-1 and PECAM-1; additional loss of vWF was accompanied by enhanced expression of caveolin-1 in SMC. Thus, associated flow-induced shear stress or inflammation, but not elevated pulmonary artery pressure alone, disrupts endothelial caveolin-1. Subsequent vWF loss, indicative of extensive endothelial damage is associated with enhanced expression of caveolin-1 in SMC, which may worsen the disease.