Aging Increases Hypoxia-Induced Endothelial Permeability and Blood-Brain Barrier Dysfunction by Upregulating Arginase-II.

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.

Elevation of Arginase-II in Podocytes Contributes to Age-Associated Albuminuria in Male Mice.

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.

Role of pulmonary epithelial arginase-II in activation of fibroblasts and lung inflammaging.

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.

Paracrine Effects of Renal Proximal Tubular Epithelial Cells on Podocyte Injury under Hypoxic Conditions Are Mediated by Arginase-II and TGF-ß1.

Hypoxia is an important risk for renal disease. The mitochondrial enzyme arginase-II (Arg-II) is expressed and/or induced by hypoxia in proximal tubular epithelial cells (PTECs) and in podocytes, leading to cellular damage. Because PTECs are vulnerable to hypoxia and located in proximity to podocytes, we examined the role of Arg-II in the crosstalk of PTECs under hypoxic conditions with podocytes. A human PTEC cell line (HK2) and a human podocyte cell line (AB8/13) were cultured. Arg-ii gene was ablated by CRISPR/Case9 in both cell types. HK2 cells were exposed to normoxia (21% O2) or hypoxia (1% O2) for 48 h. Conditioned medium (CM) was collected and transferred to the podocytes. Podocyte injuries were then analyzed. Hypoxic (not normoxic) HK2-CM caused cytoskeletal derangement, cell apoptosis, and increased Arg-II levels in differentiated podocytes. These effects were absent when arg-ii in HK2 was ablated. The detrimental effects of the hypoxic HK2-CM were prevented by TGF-ß1 type-I receptor blocker SB431542. Indeed, TGF-ß1 levels in hypoxic HK2-CM (but not arg-ii-/--HK2-CM) were increased. Furthermore, the detrimental effects of TGF-ß1 on podocytes were prevented in arg-ii-/--podocytes. This study demonstrates crosstalk between PTECs and podocytes through the Arg-II-TGF-ß1 cascade, which may contribute to hypoxia-induced podocyte damage.

Role of Arginase-II in Podocyte Injury under Hypoxic Conditions.

Hypoxia plays a crucial role in acute and chronic renal injury, which is attributable to renal tubular and glomerular cell damage. Some studies provide evidence that hypoxia-dependent upregulation of the mitochondrial enzyme arginase type-II (Arg-II) in tubular cells promotes renal tubular injury. It is, however, not known whether Arg-II is also expressed in glomerular cells, particularly podocytes under hypoxic conditions, contributing to hypoxia-induced podocyte injury. The effects of hypoxia on human podocyte cells (AB8/13) in cultures and on isolated kidneys from wild-type (wt) and arg-ii gene-deficient (arg-ii-/-) mice ex vivo, as well as on mice of the two genotypes in vivo, were investigated, respectively. We found that the Arg-II levels were enhanced in cultured podocytes in a time-dependent manner over 48 h, which was dependent on the stabilization of hypoxia-inducible factor 1α (HIF1α). Moreover, a hypoxia-induced derangement of cellular actin cytoskeletal fibers, a decrease in podocin, and an increase in mitochondrial ROS (mtROS) generation-as measured by MitoSOX-were inhibited by adenoviral-mediated arg-ii gene silencing. These effects of hypoxia on podocyte injury were mimicked by the HIFα stabilizing drug DMOG, which inhibits prolyl hydroxylases (PHD), the enzymes involved in HIFα degradation. The silencing of arg-ii prevented the detrimental effects of DMOG on podocytes. Furthermore, the inhibition of mtROS generation by rotenone-the inhibitor of respiration chain complex-I-recapitulated the protective effects of arg-ii silencing on podocytes under hypoxic conditions. Moreover, the ex vivo experiments with isolated kidney tissues and the in vivo experiments with mice exposed to hypoxic conditions showed increased Arg-II levels in podocytes and decreased podocyte markers regarding synaptopodin in wt mice but not in arg-ii-/- mice. While age-associated albuminuria was reduced in the arg-ii-/- mice, the hypoxia-induced increase in albuminuria was, however, not significantly affected in the arg-ii-/-. Our study demonstrates that Arg-II in podocytes promotes cell injury. Arg-ii ablation seems insufficient to protect mice in vivo against a hypoxia-induced increase in albuminuria, but it does reduce albuminuria in aging.

Hypoxia Induces Renal Epithelial Injury and Activates Fibrotic Signaling Through Up-Regulation of Arginase-II.

The ureohydrolase, type-II arginase (Arg-II), is a mitochondrial enzyme metabolizing L-arginine into urea and L-ornithine and is highly expressed in renal proximal tubular cells (PTC) and upregulated by renal ischemia. Recent studies reported contradictory results on the role of Arg-II in renal injury. The aim of our study is to investigate the function of Arg-II in renal epithelial cell damage under hypoxic conditions. Human renal epithelial cell line HK2 was cultured under hypoxic conditions for 12-48 h. Moreover, ex vivo experiments with isolated kidneys from wild-type (WT) and genetic Arg-II deficient mice (Arg-II-/- ) were conducted under normoxic and hypoxic conditions. The results show that hypoxia upregulates Arg-II expression in HK2 cells, which is inhibited by silencing both hypoxia-inducible factors (HIFs) HIF1α and HIF2α. Treatment of the cells with dimethyloxaloylglycine (DMOG) to stabilize HIFα also enhances Arg-II. Interestingly, hypoxia or DMOG upregulates transforming growth factor ß1 (TGFß1) levels and collagens Iα1, which is prevented by Arg-II silencing, while TGFß1-induced collagen Iα1 expression is not affected by Arg-II silencing. Inhibition of mitochondrial complex-I by rotenone abolishes hypoxia-induced reactive oxygen species (mtROS) and TGFß1 elevation in the cells. Ex vivo experiments show elevated Arg-II and TGFß1 expression and the injury marker NGAL in the WT mouse kidneys under hypoxic conditions, which is prevented in the Arg-II-/- mice. Taking together, the results demonstrate that hypoxia activates renal epithelial HIFs-Arg-II-mtROS-TGFß1-cascade, participating in hypoxia-associated renal injury and fibrosis.

Role of tubular epithelial arginase-II in renal inflammaging.

The aging kidney undergoes complex changes and is vulnerable to injury and development of chronic kidney disease (CKD) with preponderance affecting more women than men. Evidence has been presented that the type-II L-arginine:ureohydrolase, arginase-II (Arg-II) plays a role in the acceleration of aging. Arg-II is highly expressed in the kidney. However, the role of Arg-II in renal aging is not known. This study is to investigate whether Arg-II is involved in the kidney aging process dependently on sex. Arg-II level in the kidney of wild type (WT) mice is significantly elevated with aging, which is accompanied by an increase in expression of the inflammatory cytokines/chemokines, tissue macrophages, factors involved in fibrosis, and tubulointestitial fibrosis in both males and females. This renal aging phenotype is significantly suppressed in arg-II-/- mice, mainly in the females in which Arg-II level is higher than in the males. Importantly, numerous factors such as IL-1ß, MCP1, VCAM-1, and TGFß1 are mainly localized in the proximal tubular S3 segment cells expressing Arg-II in the aging kidney. In human proximal tubular cells (HK-2), TNF-α enhances adhesion molecule expression dependently on Arg-II upregulation. Overexpression of Arg-II in the cells enhances TGFß1 levels which is prevented by mitochondrial ROS inhibition. In summary, our study reveals that renal proximal tubular Arg-II plays an important role in the kidney aging process in females. Arg-II could be a promising therapeutic target for the treatment and prevention of aging-associated kidney diseases.

Arginase-II promotes melanoma migration and adhesion through enhancing hydrogen peroxide production and STAT3 signaling.

Elevated arginase type II (Arg-II) associates with higher grade tumors. Its function and underlying molecular mechanisms in melanoma remain elusive. In the present study, we observed a significantly higher frequency of Arg-II expression in melanoma of patients with metastasis than those without metastasis. Silencing Arg-II in two human melanoma cell lines slowed down the cell growth, while overexpression of native but not a catalytically inactive Arg-II promoted cell proliferation without affecting cell death. Treatment of cells with arginase inhibitor also reduced melanoma cell number, demonstrating that Arg-II promotes melanoma cell proliferation dependently of its enzymatic activity. However, results from silencing Arg-II or overexpressing native or the inactive Arg-II as well as treatment with arginase inhibitor showed that Arg-II promotes melanoma metastasis-related processes, such as melanoma cell migration and adhesion on endothelial cells, independently of its enzymatic activity. Moreover, the treatment of the cells with STAT3 inhibitor suppressed Arg-II-promoted melanoma cell migration and adhesion. Furthermore, catalase, but not superoxide dismutase, prevented STAT3 activation as well as increased melanoma cell migration and adhesion induced by overexpressing native or the inactive Arg-II. Taken together, our study uncovers both activity-dependent and independent mechanisms of Arg-II in promoting melanoma progression. While Arg-II enhances melanoma cell proliferation through polyamine dependently of its enzymatic activity, it promotes metastasis-related processes, that is, migration and adhesion onto endothelial cell, through mitochondrial H2 O2 -STAT3 pathway independently of the enzymatic activity. Suppressing Arg-II expression rather than inhibiting its enzymatic activity may, therefore, represent a novel strategy for the treatment of melanoma.

Inhibition of p38mapk Reduces Adipose Tissue Inflammation in Aging Mediated by Arginase-II.

BACKGROUND: Adipose tissue inflammation occurs not only in obesity but also in aging and is mechanistically linked with age-associated diseases. Studies show that ablation of the l-arginine-metabolizing enzyme arginase-II (Arg-II) reduces adipose tissue inflammation and improves glucose tolerance in obesity. However, the role of Arg-II in aging adipose tissue inflammation is not clear. OBJECTIVE: This study investigated the role of Arg-II in age-associated adipose tissue inflammation. METHODS: Visceral adipose tissues of young (3-6 months) and old (20-24 months) wild-type (WT) and Arg-II-/- mice were investigated. Immunofluorescence confocal microscopy was performed for analysis of macrophage accumulation and cellular localization of arginase and cytokines; expression of arginase and cytokines was analyzed by qRT-PCR or immunoblotting or ELISA; activation of mitogen-activated protein kinases in adipose tissues was analyzed by immunoblotting; and arginase activity was measured by colorimetric determination of urea production. RESULTS: In the old WT mice, there is more macrophage accumulation in the visceral adipose tissues than in Arg-II knockout animals. An age-associated increase in arginase activity and Arg-II expression in adipose tissues of WT mice is observed. Arg-II knockout enhances Arg-I expression and activity, but inhibits interleukin (IL)-6 expression and secretion and reduces active p38mapk in aging adipose tissue macrophages and stromal cells. Treatment of aging adipose tissues of WT mice with a specific p38mapk inhibitor SB203580 reduces IL-6 secretion. CONCLUSIONS: Arg-II promotes IL-6 production in aging adipose tissues through p38mapk. The results suggest that targeting Arg-II or inhibiting p38mapk could be beneficial in reducing age-associated adipose tissue inflammation.

Detrimental Effects of Chronic L-Arginine Rich Food on Aging Kidney.

The impaired L-arginine/nitric oxide pathway is a well-recognized mechanism for cardiovascular and renal diseases with aging. Therefore, supplementation of L-arginine is widely proposed to boost health or as adjunct therapy for the patients. However, clinical data, show adverse effects and even enhanced mortality in patients receiving long-term L-arginine supplementation. The effects of long-term L-arginine supplementation on kidney aging and the underlying mechanisms remain elusive. Moreover, high protein and high amino acid diet has been thought detrimental for kidney. We therefore investigated effects of chronic dietary L-arginine supplementation on kidney aging. In both young (4 months) and old (18-24 months) mice, animals either receive standard chow containing 0.65% L-arginine or diet supplemented with L-arginine to 2.46% for 16 weeks. Inflammation and fibrosis markers and albuminuria are then analyzed. Age-associated increases in tnf-α, il-1ß, and il-6, vcam-1, icam-1, mcp1, inos, and macrophage infiltration, collagen expression, and S6K1 activation are observed, which is not favorably affected, but rather further enhanced, by L-arginine supplementation. Importantly, L-arginine supplementation further enhances age-associated albuminuria and mortality particularly in females, accompanied by elevated renal arginase-II (Arg-II) levels. The enhanced albuminuria by L-arginine supplementation in aging is not protected in Arg-II-/- mice. In contrast, L-arginine supplementation increases ROS and decreases nitric oxide production in old mouse aortas, which is reduced in Arg-II-/- mice. The results do not support benefits of long-term L-arginine supplementation. It rather accelerates functional decline of kidney and vasculature in aging. Thus, the long-term dietary L-arginine supplementation should be avoided particularly in elderly population.

Hypoxia Enhances Endothelial Intercellular Adhesion Molecule 1 Protein Level Through Upregulation of Arginase Type II and Mitochondrial Oxidative Stress.

Hypoxia plays a crucial role in the pathogenesis of cardiovascular diseases. Mitochondrial enzyme arginase type II (Arg-II) is reported to lead to endothelial dysfunction and enhance the expression of endothelial inflammatory adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). In this study, we investigate the role of Arg-II in hypoxia-induced endothelial activation and the potential underlying mechanisms. Exposure of the human endothelial cells to hypoxia induced a time-dependent increase in Arg-II, HIF1α, HIF2α, and ICAM-1 protein level, whereas no change in the protein level of VCAM-1 and E-selectin was observed. Similar effects were obtained in cells treated with a hypoxia mimetic Dimethyloxaloylglycine (DMOG). Silencing HIF1α, but not HIF2α, reversed hypoxia-induced upregulation of Arg-II. Moreover, silencing Arg-II prevented the ICAM-1 upregulation induced by hypoxia or DMOG. Furthermore, the endothelial cells incubated under hypoxic condition or treated with DMOG or hypoxia enhanced monocyte adhesion, which was inhibited by silencing Arg-II. Lastly, silencing Arg-II prevented hypoxia-induced mitochondrial superoxide production in endothelial cells, and hypoxia-induced ICAM-1 upregulation was reversed by mitochondrial electron transport inhibitor rotenone. These data demonstrate that hypoxia enhances ICAM-1 protein level and monocyte-endothelial interaction through HIF1α-mediated increase in Arg-II protein level on leading to increased mitochondrial reactive oxygen species production. These effects of hypoxia on endothelial cells may play a key role in cardiovascular diseases. Our results suggest that Arg-II could be a promising therapeutic target to prevent hypoxia-induced vascular damage/dysfunction.

Myosin 1b Regulates Nuclear AKT Activation by Preventing Localization of PTEN in the Nucleus.

Insulin-induced AKT activation is dependent on phosphoinositide 3-kinase and opposed by tumor suppressor phosphatase and tensin homolog (PTEN). Our previous study demonstrates that myosin 1b (MYO1B) mediates arginase-II-induced activation of mechanistic target of rapamycin complex 1 that is regulated by AKT. However, the role of MYO1B in AKT activation is unknown. Here we show that silencing MYO1B in mouse embryonic fibroblasts (MEF) inhibits insulin-induced nuclear but not cytoplasmic AKT activation accompanied by elevated nuclear PTEN level. Co-immunoprecipitation, co-immunostaining, and proximity ligation assay show an interaction of MYO1B and PTEN resulting in reduced nuclear PTEN. Moreover, the elevated nuclear PTEN upon silencing MYO1B promotes apoptosis of MEFs and melanoma B16F10 cells. Taken together, we demonstrate that MYO1B, by interacting with PTEN, prevents nuclear localization of PTEN contributing to nuclear AKT activation and suppression of cell apoptosis. This may present a therapeutic approach for cancer treatment such as melanoma.

Arginase-II negatively regulates renal aquaporin-2 and water reabsorption.

Type-II l-arginine:ureahydrolase, arginase-II (Arg-II), is abundantly expressed in the kidney. The physiologic role played by Arg-II in the kidney remains unknown. Herein, we report that in mice that are deficient in Arg-II (Arg-II-/-), total and membrane-associated aquaporin-2 (AQP2) protein levels were significantly higher compared with wild-type (WT) controls. Water deprivation enhanced Arg-II expression, AQP2 levels, and membrane association in collecting ducts. Effects of water deprivation on AQP2 were stronger in Arg-II-/- mice than in WT mice. Accordingly, a decrease in urine volume and an increase in urine osmolality under water deprivation were more pronounced in Arg-II-/- mice than in WT mice, which correlated with a weaker increase in plasma osmolality in Arg-II-/- mice. There was no difference in vasopressin release under water deprivation conditions between either genotype of mice. Although total AQP2 and phosphorylated AQP2-S256 levels (mediated by PKA) in kidneys under water deprivation conditions were significantly higher in Arg-II-/- mice compared with WT animals, there is no difference in the ratio of AQP2-S256:AQP2. In cultured mouse collecting duct principal mCCDcl1 cells, expression of both Arg-II and AQP2 were enhanced by the vasopressin type 2 receptor agonist, desamino- d-arginine vasopressin (dDAVP). Silencing Arg-II enhanced the expression and membrane association of AQP2 by dDAVP without influencing cAMP levels. In conclusion, in vivo and in vitro experiments demonstrate that Arg-II negatively regulates AQP2 and the urine-concentrating capability in kidneys via a mechanism that is not associated with the modulation of the cAMP pathway.-Huang, J., Montani, J.-P., Verrey, F., Feraille, E., Ming, X.-F., Yang, Z. Arginase-II negatively regulates renal aquaporin-2 and water reabsorption.

Arginase-II activates mTORC1 through myosin-1b in vascular cell senescence and apoptosis.

Type-II L-arginine:ureahydrolase, arginase-II (Arg-II), is shown to activate mechanistic target of rapamycin complex 1 (mTORC1) pathway and contributes to cell senescence and apoptosis. In an attempt to elucidate the underlying mechanism, we identified myosin-1b (Myo1b) as a mediator. Overexpression of Arg-II induces re-distribution of lysosome and mTOR but not of tuberous sclerosis complex (TSC) from perinuclear area to cell periphery, dissociation of TSC from lysosome and activation of mTORC1-ribosomal protein S6 kinase 1 (S6K1) pathway. Silencing Myo1b prevents all these alterations induced by Arg-II. By overexpressing Myo1b or its mutant with point mutation in its pleckstrin homology (PH) domain we further demonstrate that this effect of Myo1b is dependent on its PH domain that is required for Myo1b-lysosome association. Notably, Arg-II promotes association of Myo1b with lysosomes. In addition, we show that in senescent vascular smooth muscle cells with elevated endogenous Arg-II, silencing Myo1b prevents Arg-II-mediated lysosomal positioning, dissociation of TSC from lysosome, mTORC1 activation and cell apoptosis. Taken together, our study demonstrates that Myo1b mediates the effect of Arg-II in activating mTORC1-S6K1 through promoting peripheral lysosomal positioning, that results in spatial separation and thus dissociation of TSC from lysosome, leading to hyperactive mTORC1-S6K1 signaling linking to cellular senescence/apoptosis.

Kidney Mass Reduction Leads to l-Arginine Metabolism-Dependent Blood Pressure Increase in Mice.

BACKGROUND: Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of l-arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts l-arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. METHODS AND RESULTS: In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post-UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. l-citrulline, that is used in the kidney to produce l-arginine, was elevated post-UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with l-arginine and is a marker for renal failure. Interestingly, the UNX-induced blood pressure increase was prevented by supplementation of the diet with 5% of the l-arginine precursor, l-citrulline. Because l-arginine is metabolized in the kidney and other peripheral tissues by arginase-2, we tested whether the lack of this metabolic pathway also compensates for decreased l-arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase-2 knockout mice (Arg-2-/-) neither displayed an increase in asymmetric dimethylarginine and l-citrulline plasma levels nor a significant increase in blood pressure. CONCLUSIONS: UNX leads to a small increase in blood pressure that is prevented by l-citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on l-arginine metabolism.

Arginase-II Deficiency Extends Lifespan in Mice.

The mitochondrial arginase type II (Arg-II) has been shown to interact with ribosomal protein S6 kinase 1 (S6K1) and mitochondrial p66Shc and to promote cell senescence, apoptosis and inflammation under pathological conditions. However, the impact of Arg-II on organismal lifespan is not known. In this study, we demonstrate a significant lifespan extension in mice with Arg-II gene deficiency (Arg-II-/-) as compared to wild type (WT) control animals. This effect is more pronounced in the females than in the males. The gender difference is associated with higher Arg-II expression levels in the females than in the males in skin and heart at both young and old age. Ablation of Arg-II gene significantly reduces the aging marker p16INK4a levels in these tissues of old female mice, whereas in the male mice this effect of Arg-II deficiency is weaker. In line with this observation, age-associated increases in S6K1 signaling and p66Shc levels in heart are significantly attenuated in the female Arg-II-/- mice. In the male mice, only p66Shc but not S6K1 signaling is reduced. In summary, our study demonstrates that Arg-II may play an important role in the acceleration of aging in mice. Genetic disruption of Arg-II in mouse extends lifespan predominantly in females, which relates to inhibition of S6K1, p66Shc, and p16INK4a. Thus, Arg-II may represent a promising target to decelerate aging process and extend lifespan as well as to treat age-related diseases.

Arginase-II Promotes Tumor Necrosis Factor-α Release From Pancreatic Acinar Cells Causing ß-Cell Apoptosis in Aging.

Aging is associated with glucose intolerance. Arginase-II (Arg-II), the type-II L-arginine-ureahydrolase, is highly expressed in pancreas. However, its role in regulation of pancreatic ß-cell function is not known. Here we show that female (not male) mice deficient in Arg-II (Arg-II-/-) are protected from age-associated glucose intolerance and reveal greater glucose induced-insulin release, larger islet size and ß-cell mass, and more proliferative and less apoptotic ß-cells compared with the age-matched wild-type (WT) controls. Moreover, Arg-II is mainly expressed in acinar cells and is upregulated with aging, which enhances p38 mitogen-activated protein kinase (p38 MAPK) activation and release of tumor necrosis factor-α (TNF-α). Accordingly, conditioned medium of isolated acinar cells from old WT (not Arg-II-/-) mice contains higher TNF-α levels than the young mice and stimulates ß-cell apoptosis and dysfunction, which are prevented by a neutralizing anti-TNF-α antibody. In acinar cells, our study demonstrates an age-associated Arg-II upregulation, which promotes TNF-α release through p38 MAPK leading to ß-cell apoptosis, insufficient insulin secretion, and glucose intolerance in female rather than male mice.

Arginase-I enhances vascular endothelial inflammation and senescence through eNOS-uncoupling.

BACKGROUND: Augmented arginase-II (Arg-II) is implicated in endothelial senescence and inflammation through a mutual positive regulatory circuit with S6K1. This study was conducted to investigate whether Arg-I, another isoform of arginase that has been also reported to play a role in vascular endothelial dysfunction, promotes endothelial senescence through similar mechanisms. RESULTS: The non-senescent human endothelial cells from umbilical veins (passage 2 to 4) were transduced with empty recombinant adenovirus vector (rAd/CMV) as control or rAd/CMV-Arg-I to overexpress Arg-I. Overexpressing Arg-I promoted eNOS-uncoupling, enhanced senescence markers including p53-S15, p21 and senescence-associated ß-galactosidase (SA-ß-gal) staining, and increased inflammatory vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) as well as monocyte adhesion to endothelial cells without activating S6K1. All the effects of Arg-I were inhibited by the anti-oxidant N-acetylcysteine (NAC). CONCLUSIONS: Our study demonstrates that Arg-I promotes endothelial senescence and inflammatory responses through eNOS-uncoupling unrelated to activation of the S6K1 pathway.

Genetic Targeting of Arginase-II in Mouse Prevents Renal Oxidative Stress and Inflammation in Diet-Induced Obesity.

Obesity is associated with development and progression of chronic kidney disease (CKD). Recent evidence demonstrates that enhanced levels of the L-arginine:ureahydrolase, including the two isoenzymes arginase-I (Arg-I) and arginase-II (Arg-II) in vascular endothelial cells promote uncoupling of endothelial nitric oxide synthase (eNOS), leading to increased superoxide radical anion and decreased NO production thereby endothelial dysfunction. Arg-II but not Arg-I is abundantly expressed in kidney and the role of Arg-II in CKD is uncertain and controversial. We aimed to investigate the role of Arg-II in renal damage associated with diet-induced obesity mouse model. Wild type (WT) C57BL/6 mice and mice deficient in Arg-II gene (Arg-II-/-) were fed with either a normal chow (NC) or a high-fat-diet (HFD) for 14 weeks (starting at the age of 7 weeks) to induce obesity. In WT mice, HFD feeding caused frequent renal lipid accumulation, enhancement of renal reactive oxygen species (ROS) levels which could be attenuated by a NOS inhibitor, suggesting uncoupling of NOS in kidney. HFD feeding also significantly augmented renal Arg-II expression and activity. All the alterations in the kidney under HFD feeding were reduced in Arg-II-/- mice. Moreover, mesangial expansion as analyzed by Periodic Acid Schiff (PAS) staining and renal expression of vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HFD-fed WT mouse assessed by immunoblotting were reduced in the HFD-fed Arg-II-/- mice, although there was no significant difference in body weight and renal weight/body weight ratio between the WT and Arg-II-/- mice. Thus, Arg-II expression/activity is enhanced in kidney of diet-induced obesity mice. Genetic targeting of Arg-II prevents renal damage associated with obesity, suggesting an important role of Arg-II in obesity-associated renal disease development.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries.

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2(.-)) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2(.-) levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2(.-), it is difficult to measure NO and O2(.-) directly in a blood vessel. Numerous methods have been developed to measure NO and O2(.-) production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2(.-) using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2(.-) levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2(.-) in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.