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Increased endothelial permeability plays an important role in blood-brain barrier (BBB) dysfunction and is implicated in neuronal injury in many diseased conditions. BBB disruption is primarily determined by dysfunction of endothelial cell-cell junctions. Deprivation of oxygen supply or hypoxia, a common feature of a variety of human diseases, is a major risk factor for BBB disruption. The molecular regulatory mechanisms of hypoxia-induced BBB dysfunction remain incompletely understood. The mitochondrial enzyme, arginase type II (Arg-II), has been shown to promote endothelial dysfunction. However, its role in hypoxia-induced BBB dysfunction has not been explored. In the C57BL/6J mouse model, hypoxia (8% O2, 24 hours) augments vascular Arg-II in the hippocampus, decreases cell-cell junction protein levels of Zonula occludens-1 (ZO-1), occludin, and CD31 in endothelial cells, increases BBB leakage in the brain in old mice (20 to 24 months) but not in young animals (3 to 6 months). These effects of hypoxia in aging are suppressed in arg-ii-/- mice. Moreover, the age-associated vulnerability of endothelial integrity to hypoxia is demonstrated in senescent human brain microvascular endothelial cell (hCMEC/D3) culture model. Further results in the cell culture model show that hypoxia augments Arg-II, decreases ZO-1 and occludin levels, and increases endothelial permeability, which is prevented by arg-ii gene silencing or by inhibition of mitochondrial reactive oxygen species (mtROS) production. Our study demonstrates an essential role of Arg-II in increased endothelial permeability and BBB dysfunction by promoting mtROS generation, resulting in decreased endothelial cell-cell junction protein levels under hypoxic conditions particularly in aging.

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One of the manifestations of renal aging is podocyte dysfunction and loss, which are associated with proteinuria and glomerulosclerosis. Studies show a male bias in glomerular dysfunction and chronic kidney diseases, and the underlying mechanisms remain obscure. Recent studies demonstrate the role of an age-associated increase in arginase-II (Arg-II) in proximal tubules of both male and female mice. However, it is unclear whether Arg-II is also involved in aging glomeruli. The current study investigates the role of the sex-specific elevation of Arg-II in podocytes in age-associated increased albuminuria. Young (3-4 months) and old (20-22 months) male and female mice of wt and arginase-II knockout (arg-ii-/-) were used. Albuminuria was employed as a readout of glomerular function. Cellular localization and expression of Arg-II in glomeruli were analyzed using an immunofluorescence confocal microscope. A more pronounced age-associated increase in albuminuria was found in male than in female mice. An age-associated induction of Arg-II in glomeruli and podocytes (as demonstrated by co-localization of Arg-II with the podocyte marker synaptopodin) was also observed in males but not in females. Ablation of the arg-ii gene in mice significantly reduces age-associated albuminuria in males. Also, age-associated decreases in podocyte density and glomerulus hypertrophy are significantly prevented in male arg-ii-/- but not in female mice. However, age-associated glomerulosclerosis is not affected by arg-ii ablation in both sexes. These results demonstrate a role of Arg-II in sex-specific podocyte injury in aging. They may explain the sex-specific differences in the development of renal disease in humans during aging.

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Elevated arginases including type-I (Arg-I) and type-II isoenzyme (Arg-II) are reported to play a role in aging, age-associated organ inflammaging, and fibrosis. A role of arginase in pulmonary aging and underlying mechanisms are not explored. Our present study shows increased Arg-II levels in aging lung of female mice, which is detected in bronchial ciliated epithelium, club cells, alveolar type 2 (AT2) pneumocytes, and fibroblasts (but not vascular endothelial and smooth muscle cells). Similar cellular localization of Arg-II is also observed in human lung biopsies. The age-associated increase in lung fibrosis and inflammatory cytokines, including IL-1ß and TGF-ß1 that are highly expressed in bronchial epithelium, AT2 cells, and fibroblasts, are ameliorated in arg-ii deficient (arg-ii-/- ) mice. The effects of arg-ii-/- on lung inflammaging are weaker in male as compared to female animals. Conditioned medium (CM) from human Arg-II-positive bronchial and alveolar epithelial cells, but not that from arg-ii-/- cells, activates fibroblasts to produce various cytokines including TGF-ß1 and collagen, which is abolished by IL-1ß receptor antagonist or TGF-ß type I receptor blocker. Conversely, TGF-ß1 or IL-1ß also increases Arg-II expression. In the mouse models, we confirmed the age-associated increase in IL-1ß and TGF-ß1 in epithelial cells and activation of fibroblasts, which is inhibited in arg-ii-/- mice. Taken together, our study demonstrates a critical role of epithelial Arg-II in activation of pulmonary fibroblasts via paracrine release of IL-1ß and TGF-ß1, contributing to pulmonary inflammaging and fibrosis. The results provide a novel mechanistic insight in the role of Arg-II in pulmonary aging.

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During physical exercise or stress, the sympathetic system stimulates cardiac contractility via ß-adrenergic receptor (ß-AR) activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent "hyperphosphorylation" of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation-contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch-clamp recordings and confocal Ca2+ imaging during ß-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (I Ca) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during ß-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

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