Calbindin 2-specific deletion of arginase 2 preserves visual function after optic nerve crush.

We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2cre/+ A2 f/f), myeloid-specific A2 KO (LysMcre/+ A2f/f), endothelial-specific A2 KO (Cdh5cre/+ A2f/f), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1ß, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility.

BACKGROUND & AIMS: Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. METHODS: Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. RESULTS: Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. CONCLUSIONS: Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Role of acyl-coenzyme A: cholesterol transferase 1 (ACAT1) in retinal neovascularization.

BACKGROUND: We have investigated the efficacy of a new strategy to limit pathological retinal neovascularization (RNV) during ischemic retinopathy by targeting the cholesterol metabolizing enzyme acyl-coenzyme A: cholesterol transferase 1 (ACAT1). Dyslipidemia and cholesterol accumulation have been strongly implicated in promoting subretinal NV. However, little is known about the role of cholesterol metabolism in RNV. Here, we tested the effects of inhibiting ACAT1 on pathological RNV in the mouse model of oxygen-induced retinopathy (OIR). METHODS: In vivo studies used knockout mice that lack the receptor for LDL cholesterol (LDLR-/-) and wild-type mice. The wild-type mice were treated with a specific inhibitor of ACAT1, K604 (10 mg/kg, i.p) or vehicle (PBS) during OIR. In vitro studies used human microglia exposed to oxygen-glucose deprivation (OGD) and treated with the ACAT1 inhibitor (1 µM) or PBS. RESULTS: Analysis of OIR retinas showed that increased expression of inflammatory mediators and pathological RNV were associated with significant increases in expression of the LDLR, increased accumulation of neutral lipids, and formation of toxic levels of cholesterol ester (CE). Deletion of the LDLR completely blocked OIR-induced RNV and significantly reduced the AVA. The OIR-induced increase in CE formation was accompanied by significant increases in expression of ACAT1, VEGF and inflammatory factors (TREM1 and MCSF) (p < 0.05). ACAT1 was co-localized with TREM1, MCSF, and macrophage/microglia makers (F4/80 and Iba1) in areas of RNV. Treatment with K604 prevented retinal accumulation of neutral lipids and CE formation, inhibited RNV, and decreased the AVA as compared to controls (p < 0.05). The treatment also blocked upregulation of LDLR, ACAT1, TREM1, MCSF, and inflammatory cytokines but did not alter VEGF expression. K604 treatment of microglia cells also blocked the effects of OGD in increasing expression of ACAT1, TREM1, and MCSF without altering VEGF expression. CONCLUSIONS: OIR-induced RNV is closely associated with increases in lipid accumulation and CE formation along with increased expression of LDLR, ACAT1, TREM1, and MCSF. Inhibiting ACAT1 blocked these effects and limited RNV independently of alterations in VEGF expression. This pathway offers a novel strategy to limit vascular injury during ischemic retinopathy.

Deficiency of the novel high mobility group protein HMGXB4 protects against systemic inflammation-induced endotoxemia in mice.

Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.

Dichotomous Role of Tumor Necrosis Factor in Pulmonary Barrier Function and Alveolar Fluid Clearance.

Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF's anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS.

Mechanisms of obesity-induced metabolic and vascular dysfunctions.

Obesity has reached epidemic proportions and its prevalence is climbing. Obesity is characterized by hypertrophied adipocytes with a dysregulated adipokine secretion profile, increased recruitment of inflammatory cells, and impaired metabolic homeostasis that eventually results in the development of systemic insulin resistance, a phenotype of type 2 diabetes. Nitric oxide synthase (NOS) is an enzyme that converts L-arginine to nitric oxide (NO), which functions to maintain vascular and adipocyte homeostasis. Arginase is a ureohydrolase enzyme that competes with NOS for L-arginine. Arginase activity/expression is upregulated in obesity, which results in diminished bioavailability of NO, impairing both adipocyte and vascular endothelial cell function. Given the emerging role of NO in the regulation of adipocyte physiology and metabolic capacity, this review explores the interplay between arginase and NO, and their effect on the development of metabolic disorders, cardiovascular diseases, and mitochondrial dysfunction in obesity. A comprehensive understanding of the mechanisms involved in the development of obesity-induced metabolic and vascular dysfunction is necessary for the identification of more effective and tailored therapeutic avenues for their prevention and treatment.

Role of Arginase 2 in Systemic Metabolic Activity and Adipose Tissue Fatty Acid Metabolism in Diet-Induced Obese Mice.

Visceral adipose tissue (VAT) inflammation and metabolic dysregulation are key components of obesity-induced metabolic disease. Upregulated arginase, a ureahydrolase enzyme with two isoforms (A1-cytosolic and A2-mitochondrial), is implicated in pathologies associated with obesity and diabetes. This study examined A2 involvement in obesity-associated metabolic and vascular disorders. WT and globally deleted A2(-/-) or A1(+/-) mice were fed either a high fat/high sucrose (HFHS) diet or normal diet (ND) for 16 weeks. Increases in body and VAT weight of HFHS-fed WT mice were abrogated in A2-/-, but not A1+/-, mice. Additionally, A2-/- HFHS-fed mice exhibited higher energy expenditure, lower blood glucose, and insulin levels compared to WT HFHS mice. VAT and adipocytes from WT HFHS fed mice showed greater A2 expression and adipocyte size and reduced expression of PGC-1α, PPAR-γ, and adiponectin. A2 deletion blunted these effects, increased levels of active AMPK-α, and upregulated genes involved in fatty acid metabolism. A2 deletion prevented HFHS-induced VAT collagen deposition and inflammation, which are involved in adipocyte metabolic dysfunction. Endothelium-dependent vasorelaxation, impaired by HFHS diet, was significantly preserved in A2-/- mice, but more prominently maintained in A1+/- mice. In summary, A2 is critically involved in HFHS-induced VAT inflammation and metabolic dysfunction.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.

Obesity-induced vascular dysfunction and arterial stiffening requires endothelial cell arginase 1.

AIMS: Elevation of arginase activity has been linked to vascular dysfunction in diabetes and hypertension by a mechanism involving decreased nitric oxide (NO) bioavailability due to L-arginine depletion. Excessive arginase activity also can drive L-arginine metabolism towards the production of ornithine, polyamines, and proline, promoting proliferation of vascular smooth muscle cells and collagen formation, leading to perivascular fibrosis. We hypothesized that there is a specific involvement of arginase 1 expression within the vascular endothelial cells in this pathology. METHODS AND RESULTS: To test this proposition, we used models of type 2 diabetes and metabolic syndrome. Studies were performed using wild type (WT), endothelial-specific arginase 1 knockout (EC-A1-/-) and littermate controls(A1con) mice fed high fat-high sucrose (HFHS) or normal diet (ND) for 6 months and isolated vessels exposed to palmitate-high glucose (PA/HG) media. Some WT mice or isolated vessels were treated with an arginase inhibitor, ABH [2-(S)-amino-6-boronohexanoic acid. In WT mice, the HFHS diet promoted increases in body weight, fasting blood glucose, and post-prandial insulin levels along with arterial stiffening and fibrosis, elevated blood pressure, decreased plasma levels of L-arginine, and elevated L-ornithine. The HFHS diet or PA/HG treatment also induced increases in vascular arginase activity along with oxidative stress, reduced vascular NO levels, and impaired endothelial-dependent vasorelaxation. All of these effects except obesity and hypercholesterolemia were prevented or significantly reduced by endothelial-specific deletion of arginase 1 or ABH treatment. CONCLUSION: Vascular dysfunctions in diet-induced obesity are prevented by deletion of arginase 1 in vascular endothelial cells or arginase inhibition. These findings indicate that upregulation of arginase 1 expression/activity in vascular endothelial cells has an integral role in diet-induced cardiovascular dysfunction and metabolic syndrome.

δPKC interaction with the d subunit of F1Fo ATP synthase impairs energetics and exacerbates ischemia/reperfusion injury in isolated rat hearts.

Previously, we demonstrated protection against hypoxic injury in neonatal cardiac myocytes and reduced release of cardiac troponin I from perfused rat hearts by a novel peptide inhibitor [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVCAGRKLALKTIDWVSFDYKDDDDK-] of the delta protein kinase C (δPKC) interaction with the "d" subunit of mitochondrial F1Fo ATP synthase (dF1Fo). This peptide was developed in our laboratory and contains: an HIV-Tat protein transduction domain; a mitochondrial targeting motif; the δPKC-dF1Fo inhibitor sequence; and a FLAG epitope. In the present study the δPKC-dF1Fo inhibitor attenuated co-immunoprecipitation of δPKC with dF1Fo, improved recovery of contractility, diminished levels of tissue t-carbonyls and 4-hydroxy-2-nonenal (HNE), and reduced 2,3,5-triphenyltetrazolium chloride-monitored infarct size following simulated global ischemia/reperfusion (IR) exposures. Perfusion of hearts with this peptide prior to IR enhanced ATP levels 2.1-fold, improved ADP (state 3)- and FCCP (maximal)-stimulated respiration in mitochondrial oxygen consumption assays, and attenuated Ca(++)-induced mitochondrial swelling following ischemic injury. Mitochondrial membrane potential (assessed by JC-1) was also improved 1.6-fold by the inhibitor in hearts subsequently exposed to IR injury. Brief IR exposures did not cause mitochondrial loss of cytochrome c in the presence or absence of the inhibitor. Additionally, the inhibitor did not modify accumulation of the autophagy marker LC3II after brief IR injury. Our results support the potential for this first-in-class peptide as a translational agent for combating cardiac IR injury.

Requirement of NOX2 expression in both retina and bone marrow for diabetes-induced retinal vascular injury.

OBJECTIVE: Diabetic retinopathy, a major cause of blindness, is characterized by increased expression of vascular endothelial growth factor (VEGF), leukocyte attachment to the vessel walls and increased vascular permeability. Previous work has shown that reactive oxygen species (ROS) produced by the superoxide generating enzyme NOX2/NADPH oxidase play a crucial role in the vascular pathology. The aim of this work was to identify the cellular sources of the damaging NOX2 activity by studies using bone marrow chimera mice. METHODS: Bone marrow cells were collected from the femurs and tibias of wild type and NOX2 deficient (NOX2(-/-)) donor mice and injected intravenously into lethally irradiated NOX2(-/-) and wild type recipients. Following recovery from radiation, mice were rendered diabetic by streptozotocin injections. The following groups of bone marrow chimeras were studied: non-diabetic WT → WT, diabetic WT → WT, diabetic WT → NOX2(-/-), diabetic NOX2(-/-) → WT. After 4 weeks of diabetes, early signs of retinopathy were examined by measuring ROS, expression of VEGF and ICAM-1, leukocyte attachment to the vessel wall and vascular permeability. RESULTS: The retinas of the diabetic WT → WT chimeras showed significant increases in ROS as compared with the non-diabetic chimeras. These diabetes-induced alterations were correlated with increases in expression of VEGF and ICAM-1, leukocyte adhesion and vascular permeability. Each of these diabetes-induced alterations were significantly attenuated in the diabetic WT → NOX2(-/-) and NOX2(-/-) → WT chimera groups (p<0.05). CONCLUSION: NOX2-generated ROS produced by both bone marrow-derived cells and resident retinal cells contribute importantly to retinal vascular injury in the diabetic retina. Targeting NOX2 in bone marrow and/or retinal cells may represent a novel therapeutic strategy for the treatment/prevention of vascular injury in the diabetic retina.

Arginase 1: an unexpected mediator of pulmonary capillary barrier dysfunction in models of acute lung injury.

The integrity of epithelial and endothelial barriers in the lower airspaces of the lungs has to be tightly regulated, in order to prevent leakage and to assure efficient gas exchange between the alveoli and capillaries. Both G(-) and G(+) bacterial toxins, such as lipopolysaccharide and pneumolysin, respectively, can be released in high concentrations within the pulmonary compartments upon antibiotic treatment of patients suffering from acute respiratory distress syndrome (ARDS) or severe pneumonia. These toxins are able to impair endothelial barrier function, either directly, or indirectly, by induction of pro-inflammatory mediators and neutrophil sequestration. Toxin-induced endothelial hyperpermeability can involve myosin light chain phosphorylation and/or microtubule rearrangement. Endothelial nitric oxide synthase (eNOS) was proposed to be a guardian of basal barrier function, since eNOS knock-out mice display an impaired expression of inter-endothelial junction proteins and as such an increased vascular permeability, as compared to wild type mice. The enzyme arginase, the activity of which can be regulated by the redox status of the cell, exists in two isoforms - arginase 1 (cytosolic) and arginase 2 (mitochondrial) - both of which can be expressed in lung microvascular endothelial cells. Upon activation, arginase competes with eNOS for the substrate l-arginine, as such impairing eNOS-dependent NO generation and promoting reactive oxygen species generation by the enzyme. This mini-review will discuss recent findings regarding the interaction between bacterial toxins and arginase during acute lung injury and will as such address the role of arginase in bacterial toxin-induced pulmonary endothelial barrier dysfunction.

Hyperoxia causes regression of vitreous neovascularization by downregulating VEGF/VEGFR2 pathway.

PURPOSE: Neovascularization (NV) is a sight-threatening complication of retinal ischemia in diabetes, retinal vein occlusion, and retinopathy of prematurity. Current treatment modalities, including laser photocoagulation and repeated intraocular injection of VEGF antagonists, are invasive and not always effective, and may carry side effects. We studied the use of hyperoxia as an alternative therapeutic strategy for regressing established vitreous NV in a mouse model of oxygen-induced ischemic retinopathy. METHODS: Hyperoxia treatment (HT, 75% oxygen) was initiated on postnatal day (P)17 after the onset of vitreous NV. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to apoptosis, and expression of VEGFR2 and inflammatory molecules. Effects of intravitreal injections of VEGF-A, VEGF-E, PlGF-1, and VEGF trap were also studied. RESULTS: HT selectively reduced NV by 70% within 24 hours. It robustly increased the level of cleaved caspase-3 in the vitreous NV between 6 and 18 hours and promoted infiltration of macrophage/microglial cells. The HT-induced apoptosis was preceded by a significant reduction in VEGFR2 expression within the NV and an increase in VEGFR2 within the surrounding neural tissue. Intravitreal VEGF-A and VEGF-E (VEGFR2 agonist) but not PlGF-1 (VEGFR1 agonist) prevented HT-induced apoptosis and regression of NV. In contrast, VEGF trap and VEGFR2 blockers mimicked the effect of HT. However, intravitreal VEGF trap induced increases in inflammatory molecules while HT did not have such unwanted effect. CONCLUSIONS: HT may be clinically useful to specifically treat proliferative NV in ischemic retinopathy.

l-Citrulline Protects from Kidney Damage in Type 1 Diabetic Mice.

RATIONALE: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of l-arginine (l-arg), the substrate for endothelial nitric oxide synthase (eNOS), failed to improve vascular function. l-Citrulline (l-cit) supplementation not only increases l-arg synthesis, but also inhibits cytosolic arginase I, a competitor of eNOS for the use of l-arg, in the vasculature. AIMS: To investigate whether l-cit treatment reduces DN in streptozotocin (STZ)-induced type 1 diabetes (T1D) in mice and rats and to study its effects on arginase II (ArgII) function, the main renal isoform. METHODS: STZ-C57BL6 mice received l-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and l-cit-treated STZ-rats were evaluated. RESULTS: l-Citrulline exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis, and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, l-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 weeks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater blood urea nitrogen levels, hypertrophy, and dilated tubules than diabetic wild type (WT) mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic WT animals. l-Cit also restored nitric oxide/reactive oxygen species balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, l-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1ß and IL-12(p70) generation in the human proximal tubular cells. CONCLUSION: l-Citrulline supplementation established an anti-inflammatory profile and significantly preserved the nephron function during T1D.

Protein kinase C-α and arginase I mediate pneumolysin-induced pulmonary endothelial hyperpermeability.

Antibiotics-induced release of the pore-forming virulence factor pneumolysin (PLY) in patients with pneumococcal pneumonia results in its presence days after lungs are sterile and is a major factor responsible for the induction of permeability edema. Here we sought to identify major mechanisms mediating PLY-induced endothelial dysfunction. We evaluated PLY-induced endothelial hyperpermeability in human lung microvascular endothelial cells (HL-MVECs) and human lung pulmonary artery endothelial cells in vitro and in mice instilled intratracheally with PLY. PLY increases permeability in endothelial monolayers by reducing stable and dynamic microtubule content and modulating VE-cadherin expression. These events, dependent upon an increased calcium influx, are preceded by protein kinase C (PKC)-α activation, perturbation of the RhoA/Rac1 balance, and an increase in myosin light chain phosphorylation. At later time points, PLY treatment increases the expression and activity of arginase in HL-MVECs. Arginase inhibition abrogates and suppresses PLY-induced endothelial barrier dysfunction by restoring NO generation. Consequently, a specific PKC-α inhibitor and the TNF-derived tonoplast intrinsic protein peptide, which blunts PLY-induced PKC-α activation, are able to prevent activation of arginase in HL-MVECs and to reduce PLY-induced endothelial hyperpermeability in mice. Arginase I (AI)(+/-)/arginase II (AII)(-/-) C57BL/6 mice, displaying a significantly reduced arginase I expression in the lungs, are significantly less sensitive to PLY-induced capillary leak than their wild-type or AI(+/+)/AII(-/-) counterparts, indicating an important role for arginase I in PLY-induced endothelial hyperpermeability. These results identify PKC-α and arginase I as potential upstream and downstream therapeutic targets in PLY-induced pulmonary endothelial dysfunction.

Diabetes-induced vascular dysfunction involves arginase I.

Arginase can cause vascular dysfunction by competing with nitric oxide synthase for l-arginine and by increasing cell proliferation and collagen formation, which promote vascular fibrosis/stiffening. We have shown that increased arginase expression/activity contribute to vascular endothelial cell (EC) dysfunction. Here, we examined the roles of the two arginase isoforms, arginase I and II (AI and AII, respectively), in this process. Experiments were performed using streptozotocin-induced diabetic mice: wild-type (WT) mice and knockout mice lacking the AII isoform alone (AI(+/+)AII(-/-)) or in combination with partial deletion of AI (AI(+/-)AII (-/-)). EC-dependent vasorelaxation of aortic rings and arterial fibrosis and stiffness were assessed in relation to arginase activity and expression. Diabetes reduced mean EC-dependent vasorelaxation markedly in diabetic WT and AI(+/+)AII(-/-) aortas (53% and 44% vs. controls, respectively) compared with a 27% decrease in AI(+/-)AII (-/-) vessels. Coronary fibrosis was also increased in diabetic WT and AI(+/+)AII(-/-) mice (1.9- and 1.7-fold vs. controls, respectively) but was not altered in AI(+/-)AII (-/-) diabetic mice. Carotid stiffness was increased by 142% in WT diabetic mice compared with 51% in AI(+/+)AII(-/-) mice and 19% in AI(+/-)AII (-/-) mice. In diabetic WT and AI(+/+)AII(-/-) mice, aortic arginase activity and AI expression were significantly increased compared with control mice, but neither parameter was altered in AI(+/-)AII (-/-) mice. In summary, AI(+/-)AII (-/-) mice exhibit better EC-dependent vasodilation and less vascular stiffness and coronary fibrosis compared with diabetic WT and AI(+/+)AII(-/-) mice. These data indicate a major involvement of AI in diabetes-induced vascular dysfunction.

Neuroprotection from retinal ischemia/reperfusion injury by NOX2 NADPH oxidase deletion.

PURPOSE: The aim of this study was to determine whether NOX2, one of the homologs of NADPH oxidase, plays a role in neuronal cell death during retinal ischemia. METHODS: Ischemia reperfusion (I/R) injury was generated in C57/BL6 and NOX2(-/-) mice by increasing the intraocular pressure (IOP) to 110 mm Hg for 40 minutes followed by reperfusion. Quantitative PCR and Western blot analysis were performed to measure NOX2 expression. Reactive oxygen species (ROS) formation was assessed by dihydroethidium imaging of superoxide formation and Western blot analysis for tyrosine nitration. TUNEL assay was performed to determine cell death at 3 days after I/R. Survival of neurons within the ganglion cell layer (GCL) was assessed at 7 days after I/R by confocal morphometric imaging of retinal wholemounts immunostained with NeuN antibody. Activation of mitogen-activated protein kinases and nuclear factor κB (NF-κΒ) was measured by Western blot analysis. RESULTS: NOX2 mRNA and protein and ROS were significantly increased in wild-type I/R retinas. This effect was associated with a 60% decrease in the number of GCL neurons and a 10-fold increase in TUNEL-positive cells compared with the fellow sham control eyes. Phosphorylation of ERK and NF-κB was significantly increased in wild-type I/R retinas. Each of these effects was markedly attenuated in the NOX2(-/-) retina (P < 0.01). CONCLUSIONS: These data demonstrate that the deletion of NOX2 can reduce I/R-induced cell death and preserve retinal GCL neurons after I/R injury. The neuronal cell injury caused by I/R is associated with the activation of ERK and NF-κB signaling mechanisms.

Inflammation and diabetic retinal microvascular complications.

Diabetic retinopathy (DR) is one of the most common complications of diabetes and is a leading cause of blindness in people of the working age in Western countries. A major pathology of DR is microvascular complications such as non-perfused vessels, microaneurysms, dot/blot hemorrhages, cotton-wool spots, venous beading, vascular loops, vascular leakage and neovascularization. Multiple mechanisms are involved in these alternations. This review will focus on the role of inflammation in diabetic retinal microvascular complications and discuss the potential therapies by targeting inflammation.

Arginase 2 deletion reduces neuro-glial injury and improves retinal function in a model of retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity (ROP) is a major cause of vision impairment in low birth weight infants. While previous work has focused on defining the mechanisms of vascular injury leading to retinal neovascularization, recent studies show that neurons are also affected. This study was undertaken to determine the role of the mitochondrial arginine/ornithine regulating enzyme arginase 2 (A2) in retinal neuro-glial cell injury in the mouse model of ROP. METHODS AND FINDINGS: Studies were performed using wild type (WT) and A2 knockout (A2-/-) mice exposed to Oxygen Induced Retinopathy (OIR). Neuronal injury and apoptosis were assessed using immunohistochemistry, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end) labeling and Western blotting. Electroretinography (ERG) was used to assess retinal function. Neuro-glial injury in WT ROP mice was evident by TUNEL labeling, retinal thinning, decreases in number of rod bipolar cells and glial cell activation as compared with room air controls. Significant reduction in numbers of TUNEL positive cells, inhibition of retinal thinning, preservation of the rod bipolar cells and prevention of glial activation were observed in the A2-/- retinas. Retinal function was markedly impaired in the WT OIR mice as shown by decreases in amplitude of the b-wave of the ERG. This defect was significantly reduced in A2-/- mice. Levels of the pro-apoptotic proteins p53, cleaved caspase 9, cytochrome C and the mitochondrial protein Bim were markedly increased in WT OIR retinas compared to controls, whereas the pro-survival Mitochondrial protein BCL-xl was reduced. These alterations were largely blocked in the A2-/- OIR retina. CONCLUSIONS: Our data implicate A2 in neurodegeneration during ROP. Deletion of A2 significantly improves neuronal survival and function, possibly through the regulation of mitochondrial membrane permeability mediated apoptosis during retinal ischemia. These molecular events are associated with decreased activation of glial cells, suggesting a rescue effect on macroglia as well.

Hyperoxia therapy of pre-proliferative ischemic retinopathy in a mouse model.

PURPOSE: To investigate the therapeutic use and mechanisms of action of normobaric hyperoxia to promote revascularization and to prevent neovascularization in a mouse model of oxygen-induced ischemic retinopathy. METHODS: Hyperoxia treatment (HT, 40%-75% oxygen) was initiated on postnatal day (P) 14 during the pre-proliferative phase of ischemic retinopathy. Immunohistochemistry, ELISA, and quantitative PCR were used to assess effects on retinal vascular repair and pathologic angiogenesis in relation to glial cell injury, VEGF protein, and mRNA levels of key mediators of pathologic angiogenesis. Effects of intravitreal injections of VEGF and the VEGF inhibitor VEGFR1/Fc fusion protein were also studied. RESULTS: Administration of HT during the ischemic pre-proliferative phase of retinopathy effectively accelerated the process of revascularization while preventing the development of vitreous neovascularization. HT enhanced the formation of specialized endothelial tip cells at the edges of the repairing capillary networks and blocked the overexpression of several molecular mediators of angiogenesis, inflammation, and extracellular proteolysis. HT markedly reduced the reactive expression of GFAP in Müller cells and improved the morphology of astrocytes in the avascular region of the retina. Exogenous VEGF administered into the vitreous on P14 was not sufficient to cause vitreous neovascularization in the HT mice. Injection of the VEGF antagonist VEGFR1/Fc blocked both pathologic and physiological angiogenesis and did not rescue astrocytes. CONCLUSIONS: HT may be clinically useful to facilitate vascular repair while blocking neovascularization in the pre-proliferative stage of ischemic retinopathy by correcting a broad range of biochemical and cellular abnormalities.