In recent years, an increasing number of manuscript have been published addressing the deleterious role of arginase in endothelial dysfunction. ROS have been shown to play a crucial role in arginase activation, which in turn leads to eNOS dysfunction.

Medical College of Georgia, Augusta University

Frontiers in Immunology

Inflammation

The role of arginase in endothelial dysfunction

Rationale: Diabetic nephropathy (DN) is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of l</sc>-arginine (l</sc>-arg), the substrate for endothelial nitric oxide synthase (eNOS), failed to improve vascular function. l</sc>-Citrulline (l</sc>-cit) supplementation not only increases l</sc>-arg synthesis, but also inhibits cytosolic arginase I, a competitor of eNOS for the use of l</sc>-arg, in the vasculature.Aims: To investigate whether l</sc>-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</sc>-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and l</sc>-cit-treated STZ-rats were evaluated.Results:l</sc>-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</sc>-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</sc>-Cit also restored nitric oxide/reactive oxygen species balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, l</sc>-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1β and IL-12(p70) generation in the human proximal tubular cells.Conclusion:l</sc>-Citrulline supplementation established an anti-inflammatory profile and significantly preserved the nephron function during T1D.

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-1β 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.

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

Coronary artery disease (CAD) remains the leading cause of death in the Western societies. Diabetes mellitus (DM) is one of the highly prevalent diseases, which remarkably accelerates the development of CAD. Experimental evidence indicates that decreased bioavailability of coronary endothelial nitric oxide (NO) contributes to the development of CAD in DM. There are recent studies showing that a selective impairment of NO synthesis occurs in coronary arteries of DM patients, which is mainly due to the limited availability of endothelial NO synthase (eNOS) precursor, l</sc>-arginine. Importantly, these studies demonstrated that DM, independent of the presence of CAD, leads to selective up-regulation of arginase-1. Arginase-1 seems to play an important role in limiting l</sc>-arginine availability in the close proximity of eNOS in vessels of DM patients. This brief review examines recent clinical studies demonstrating the pathological role of vascular arginase-1 in human diabetes. Whether arginase-1, which is crucial in the synthesis of various fundamental polyamines in the body, will represent a potent therapeutic target for prevention of DM-associated CAD is still debated.

Coronary artery disease (CAD) remains the leading cause of death in the Western societies. Diabetes mellitus (DM) is one of the highly prevalent diseases, which remarkably accelerates the development of CAD. Experimental evidence indicates that decreased bioavailability of coronary endothelial nitric oxide (NO) contributes to the development of CAD in DM. There are recent studies showing that a selective impairment of NO synthesis occurs in coronary arteries of DM patients, which is mainly due to the limited availability of endothelial NO synthase (eNOS) precursor, l-arginine. Importantly, these studies demonstrated that DM, independent of the presence of CAD, leads to selective up-regulation of arginase-1. Arginase-1 seems to play an important role in limiting l-arginine availability in the close proximity of eNOS in vessels of DM patients. This brief review examines recent clinical studies demonstrating the pathological role of vascular arginase-1 in human diabetes. Whether arginase-1, which is crucial in the synthesis of various fundamental polyamines in the body, will represent a potent therapeutic target for prevention of DM-associated CAD is still debated.

Selective Up-Regulation of Arginase-1 in Coronary Arteries of Diabetic Patients

Endothelial dysfunction and resulting vascular pathology have been identified as an early hallmark of multiple diseases, including diabetes mellitus. One of the major contributors to endothelial dysfunction is a decrease in nitric oxide (NO) bioavailability, impaired NO signaling, and an increase in the amount of reactive oxygen species (ROS). In the endothelium NO is produced by endothelial nitric oxide synthase (eNOS), for which l</sc>-arginine is a substrate. Arginase, an enzyme critical in the urea cycle also metabolizes l</sc>-arginine, thereby directly competing with eNOS for their common substrate and constraining its bioavailability for eNOS, thereby compromising NO production. Arginase expression and activity is upregulated in many cardiovascular diseases including ischemia reperfusion injury, hypertension, atherosclerosis, and diabetes mellitus. More importantly, since the 1990s, specific arginase inhibitors such as N-hydroxy-guanidinium or N-hydroxy-nor-l</sc>-arginine, and boronic acid derivatives, such as, 2(S)-amino-6-boronohexanoic acid, and S-(2-boronoethyl)-l</sc>-cysteine, that can bridge the binuclear manganese cluster of arginase have been developed. These highly potent and specific inhibitors can now be used to probe arginase function and thereby modulate the redox milieu of the cell by changing the balance between NO and ROS. Inspired by this success, drug discovery programs have recently led to the identification of α–α-disubstituted amino acid based arginase inhibitors [such as (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid], that are currently under early investigation as therapeutics. Finally, some investigators concentrate on identification of plant derived compounds with arginase inhibitory capability, such as piceatannol-3′-O-β-d</sc>-glucopyranoside (PG). All of these synthesized or naturally derived small molecules may represent novel therapeutics for vascular disease particularly that associated with diabetes.

Endothelial dysfunction and resulting vascular pathology have been identified as an early hallmark of multiple diseases, including diabetes mellitus. One of the major contributors to endothelial dysfunction is a decrease in nitric oxide (NO) bioavailability, impaired NO signaling, and an increase in the amount of reactive oxygen species (ROS). In the endothelium NO is produced by endothelial nitric oxide synthase (eNOS), for which l-arginine is a substrate. Arginase, an enzyme critical in the urea cycle also metabolizes l-arginine, thereby directly competing with eNOS for their common substrate and constraining its bioavailability for eNOS, thereby compromising NO production. Arginase expression and activity is upregulated in many cardiovascular diseases including ischemia reperfusion injury, hypertension, atherosclerosis, and diabetes mellitus. More importantly, since the 1990s, specific arginase inhibitors such as N-hydroxy-guanidinium or N-hydroxy-nor-l-arginine, and boronic acid derivatives, such as, 2(S)-amino-6-boronohexanoic acid, and S-(2-boronoethyl)-l-cysteine, that can bridge the binuclear manganese cluster of arginase have been developed. These highly potent and specific inhibitors can now be used to probe arginase function and thereby modulate the redox milieu of the cell by changing the balance between NO and ROS. Inspired by this success, drug discovery programs have recently led to the identification of α–α-disubstituted amino acid based arginase inhibitors [such as (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid], that are currently under early investigation as therapeutics. Finally, some investigators concentrate on identification of plant derived compounds with arginase inhibitory capability, such as piceatannol-3′-O-β-d-glucopyranoside (PG). All of these synthesized or naturally derived small molecules may represent novel therapeutics for vascular disease particularly that associated with diabetes.

Development of Novel Arginase Inhibitors for Therapy of Endothelial Dysfunction

Overproduction of reactive oxygen species, i.e., oxidative stress, is associated with the activation of redox signaling pathways linking to inflammatory insults and cardiovascular diseases by impairing endothelial function and consequently blood flow dysregulation due to microvascular dysfunction. This review focuses on the regulation of vasomotor function in the coronary microcirculation by endothelial nitric oxide (NO) during oxidative stress and inflammation related to the activation of L-arginine consuming enzyme arginase. Superoxide produced in the vascular wall compromises vasomotor function by not only scavenging endothelium-derived NO but also inhibiting prostacyclin synthesis due to formation of peroxynitrite. The upregulation of arginase contributes to the deficiency of endothelial NO and microvascular dysfunction in various vascular diseases by initiating or following oxidative stress and inflammation. Hydrogen peroxide, a diffusible and stable oxidizing agent, exerts vasodilator function and plays important roles in the physiological regulation of coronary blood flow. In occlusive coronary ischemia, the release of hydrogen peroxide from the microvasculature helps to restore vasomotor function of coronary collateral microvessels with exercise training. However, excessive production and prolonged exposure of microvessels to hydrogen peroxide impairs NO-mediated endothelial function by reducing L-arginine availability through hydroxyl radical-dependent upregulation of arginase. The redox signaling can be a double-edged sword in the microcirculation, which helps tissue survival in one way by improving vasomotor regulation and elicits oxidative stress and tissue injury in the other way by causing vascular dysfunction. The impact of vascular arginase on the development of vasomotor dysfunction associated with angiotensin II receptor activation, hypertension, ischemia-reperfusion, hypercholesterolemia, and inflammatory insults is discussed.

Overproduction of reactive oxygen species, i.e., oxidative stress, is associated with the activation of redox signaling pathways linking to inflammatory insults and cardiovascular diseases by impairing endothelial function and consequently blood flow dysregulation due to microvascular dysfunction. This review focuses on the regulation of vasomotor function in the coronary microcirculation by endothelial nitric oxide (NO) during oxidative stress and inflammation related to the activation of l-arginine consuming enzyme arginase. Superoxide produced in the vascular wall compromises vasomotor function by not only scavenging endothelium-derived NO but also inhibiting prostacyclin synthesis due to formation of peroxynitrite. The upregulation of arginase contributes to the deficiency of endothelial NO and microvascular dysfunction in various vascular diseases by initiating or following oxidative stress and inflammation. Hydrogen peroxide, a diffusible and stable oxidizing agent, exerts vasodilator function and plays important roles in the physiological regulation of coronary blood flow. In occlusive coronary ischemia, the release of hydrogen peroxide from the microvasculature helps to restore vasomotor function of coronary collateral microvessels with exercise training. However, excessive production and prolonged exposure of microvessels to hydrogen peroxide impairs NO-mediated endothelial function by reducing l-arginine availability through hydroxyl radical-dependent upregulation of arginase. The redox signaling can be a double-edged sword in the microcirculation, which helps tissue survival in one way by improving vasomotor regulation and elicits oxidative stress and tissue injury in the other way by causing vascular dysfunction. The impact of vascular arginase on the development of vasomotor dysfunction associated with angiotensin II receptor activation, hypertension, ischemia-reperfusion, hypercholesterolemia, and inflammatory insults is discussed.

Vasomotor Regulation of Coronary Microcirculation by Oxidative Stress: Role of Arginase

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</sc>-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.

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.

Arginase 1: An Unexpected Mediator of Pulmonary Capillary Barrier Dysfunction in Models of Acute Lung Injury

Enhanced arginase (ARG) activity has been identified as a factor that reduces nitric oxide production and impairs endothelial function in vascular pathologies. Using a gene deletion model, we investigated involvement of arginase isoforms arginase 1 and 2 (ARG1 and ARG2) in hypertension and endothelial dysfunction in a mineralocorticoid-salt mouse model. Hypertension was induced in wild type (WT), partial ARG1+/− knockout (KO), and complete ARG2−/− KO mice by uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for 6-weeks. (Control uninephrectomized mice drank tap water.) After 2 weeks of DOCA-salt treatment, systolic blood pressure (SBP) was increased by ∼15 mmHg in all mouse genotypes. SBP continued to rise in DOCA-salt WT and ARG2−/− mice to ∼130 mmHg at 5–6 weeks, whereas in ARG1+/− mice SBP waned toward control levels by 6 weeks (109 ± 4 vs. 101 ± 3 mmHg, respectively). DOCA-salt treatment in WT mice increased vascular ARG activity (aorta by 1.5-fold; mesenteric artery (MA) by 2.6-fold and protein levels of ARG1 (aorta: 1.49-fold and MA: 1.73-fold) vs. WT Sham tissues. ARG2 protein increased in WT-DOCA MA (by 2.15-fold) but not in aorta compared to those of WT Sham tissues. Maximum endothelium-dependent vasorelaxation to acetylcholine was significantly reduced in DOCA-salt WT mice and largely or partially maintained in DOCA ARG1+/− and ARG2−/− mice vs. their Sham controls. DOCA-salt augmented contractile responses to phenylephrine in aorta of all mouse genotypes. Additionally, treatment of aorta or MA from WT-DOCA mice with arginase inhibitor (100 μM) improved endothelium-mediated vasorelaxation. DOCA-salt-induced coronary perivascular fibrosis (increased by 2.1-fold) in WT was prevented in ARG1+/− and reduced in ARG2−/− mice. In summary, ARG is involved in murine DOCA-salt-induced impairment of vascular function and hypertension and may represent a novel target for antihypertensive therapy.

Enhanced arginase (ARG) activity has been identified as a factor that reduces nitric oxide production and impairs endothelial function in vascular pathologies. Using a gene deletion model, we investigated involvement of arginase isoforms arginase 1 and 2 (ARG1 and ARG2) in hypertension and endothelial dysfunction in a mineralocorticoid-salt mouse model. Hypertension was induced in wild type (WT), partial ARG1+/− knockout (KO), and complete ARG2−/− KO mice by uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for 6-weeks. (Control uninephrectomized mice drank tap water.) After 2 weeks of DOCA-salt treatment, systolic blood pressure (SBP) was increased by ∼15 mmHg in all mouse genotypes. SBP continued to rise in DOCA-salt WT and ARG2−/− mice to ∼130 mmHg at 5–6 weeks, whereas in ARG1+/− mice SBP waned toward control levels by 6 weeks (109 ± 4 vs. 101 ± 3 mmHg, respectively). DOCA-salt treatment in WT mice increased vascular ARG activity (aorta by 1.5-fold; mesenteric artery (MA) by 2.6-fold and protein levels of ARG1 (aorta: 1.49-fold and MA: 1.73-fold) vs. WT Sham tissues. ARG2 protein increased in WT-DOCA MA (by 2.15-fold) but not in aorta compared to those of WT Sham tissues. Maximum endothelium-dependent vasorelaxation to acetylcholine was significantly reduced in DOCA-salt WT mice and largely or partially maintained in DOCA ARG1+/− and ARG2−/− mice vs. their Sham controls. DOCA-salt augmented contractile responses to phenylephrine in aorta of all mouse genotypes. Additionally, treatment of aorta or MA from WT-DOCA mice with arginase inhibitor (100 μM) improved endothelium-mediated vasorelaxation. DOCA-salt-induced coronary perivascular fibrosis (increased by 2.1-fold) in WT was prevented in ARG1+/− and reduced in ARG2−/− mice. In summary, ARG is involved in murine DOCA-salt-induced impairment of vascular function and hypertension and may represent a novel target for antihypertensive therapy.

Arginase 1 Mediates Increased Blood Pressure and Contributes to Vascular Endothelial Dysfunction in Deoxycorticosterone Acetate-Salt Hypertension

The endothelial production of nitric oxide (NO) mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle cell proliferation, and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS) through the conversion of its substrate, l</sc>-arginine to l</sc>-citrulline. Reduced access to l</sc>-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2) metabolize l</sc>-arginine to generate l</sc>-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of l</sc>-arginine. Indeed, supplemental l</sc>-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, this simple relationship is complicated by observations that l</sc>-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. Accordingly, the subcellular compartmentalization of intracellular l</sc>-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of pools or pockets of l</sc>-arginine. In agreement with this, there is considerable evidence supporting the importance of the subcellular localization of l</sc>-arginine metabolizing enzymes. In endothelial cells in vitro and in vivo, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localization inside the cell. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, arginosuccinate lyase, co-localize with eNOS and facilitate NO release. Herein, we highlight the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease.

The endothelial production of nitric oxide (NO) mediates endothelium-dependent vasorelaxation and restrains vascular inflammation, smooth muscle cell proliferation, and platelet aggregation. Impaired production of NO is a hallmark of endothelial dysfunction and promotes the development of cardiovascular disease. In endothelial cells, NO is generated by endothelial nitric oxide synthase (eNOS) through the conversion of its substrate, L-arginine to L-citrulline. Reduced access to L-arginine has been proposed as a major mechanism underlying reduced eNOS activity and NO production in cardiovascular disease. The arginases (Arg1 and Arg2) metabolize L-arginine to generate L-ornithine and urea and increased expression of arginase has been proposed as a mechanism of reduced eNOS activity secondary to the depletion of L-arginine. Indeed, supplemental L-arginine and suppression of arginase activity has been shown to improve endothelium-dependent relaxation and ameliorate cardiovascular disease. However, this simple relationship is complicated by observations that L-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. Accordingly, the subcellular compartmentalization of intracellular L-arginine into poorly interchangeable pools has been proposed to allow for the local depletion of pools or pockets of L-arginine. In agreement with this, there is considerable evidence supporting the importance of the subcellular localization of L-arginine metabolizing enzymes. In endothelial cells in vitro and in vivo, eNOS is found in discrete intracellular locations and the capacity to generate NO is heavily influenced by its localization inside the cell. Arg1 and Arg2 also reside in different subcellular environments and are thought to differentially influence endothelial function. The plasma membrane solute transporter, CAT-1 and the arginine recycling enzyme, arginosuccinate lyase, co-localize with eNOS and facilitate NO release. Herein, we highlight the importance of the subcellular location of eNOS and arginine transporting and metabolizing enzymes to NO release and cardiovascular disease.

The Subcellular Compartmentalization of Arginine Metabolizing Enzymes and Their Role in Endothelial Dysfunction

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased <mml:math id="M1" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>O</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo class="MathClass-punc">.</mml:mo><mml:mo class="MathClass-bin">−</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces <mml:math id="M2" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>O</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo class="MathClass-punc">.</mml:mo><mml:mo class="MathClass-bin">−</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased <mml:math id="M3" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>O</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo class="MathClass-punc">.</mml:mo><mml:mo class="MathClass-bin">−</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased <math id="M1"><msubsup><mrow><mtext>O</mtext></mrow><mrow><mn>2</mn></mrow><mrow><mo class="MathClass-punc">.</mo><mo class="MathClass-bin">−</mo></mrow></msubsup></math> formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces <math id="M2"><msubsup><mrow><mtext>O</mtext></mrow><mrow><mn>2</mn></mrow><mrow><mo class="MathClass-punc">.</mo><mo class="MathClass-bin">−</mo></mrow></msubsup></math> and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased <math id="M3"><msubsup><mrow><mtext>O</mtext></mrow><mrow><mn>2</mn></mrow><mrow><mo class="MathClass-punc">.</mo><mo class="MathClass-bin">−</mo></mrow></msubsup></math> formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

Arginase as a Mediator of Diabetic Retinopathy

Nitric oxide (NO) generated through L</sc>-arginine metabolism by endothelial nitric oxide synthase (eNOS) is an important regulator of the vessel wall. Dysregulation of this system has been implicated in various pathological vascular conditions, including atherosclerosis, angiogenesis, arteriogenesis, neointimal hyperplasia, and pulmonary hypertension. The pathophysiology involves a decreased bioavailability of NO within the vessel wall by competitive utilization of L</sc>-arginine by arginase and “eNOS uncoupling.” Generation of NO through reduction of nitrate and nitrite represents an alternative pathway that may be utilized to increase the bioavailability of NO within the vessel wall. We review the therapeutic potential of the nitrate/nitrite/NO pathway in vascular dysfunction.

Nitric oxide (NO) generated through L-arginine metabolism by endothelial nitric oxide synthase (eNOS) is an important regulator of the vessel wall. Dysregulation of this system has been implicated in various pathological vascular conditions, including atherosclerosis, angiogenesis, arteriogenesis, neointimal hyperplasia, and pulmonary hypertension. The pathophysiology involves a decreased bioavailability of NO within the vessel wall by competitive utilization of L-arginine by arginase and “eNOS uncoupling.” Generation of NO through reduction of nitrate and nitrite represents an alternative pathway that may be utilized to increase the bioavailability of NO within the vessel wall. We review the therapeutic potential of the nitrate/nitrite/NO pathway in vascular dysfunction.

Therapeutic Potential of the Nitrite-Generated NO Pathway in Vascular Dysfunction

Oxidative stress and inflammation in the vascular wall are essential mechanisms of atherosclerosis and vascular dysfunctions associated with risk factors such as metabolic diseases, aging, hypertension, etc. Evidence has been provided that activation of the vascular endothelial cells in the presence of the risk factors promotes oxidative stress and vascular inflammatory responses, leading to acceleration of atherosclerotic vascular disease. Increasing number of studies from recent years demonstrates that uncoupling of endothelial nitric oxide synthase (eNOS), whereby the enzyme eNOS produces detrimental amount of superoxide anion <mml:math id="M1" xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:msubsup><mml:mrow><mml:mtext>O</mml:mtext></mml:mrow><mml:mrow><mml:mn>2</mml:mn></mml:mrow><mml:mrow><mml:mo class="MathClass-bin">−</mml:mo></mml:mrow></mml:msubsup></mml:math></inline-formula> instead the vasoprotective nitric oxide (NO⋅), plays a critical role in vascular dysfunction under various pathophysiological conditions and in aging. The mechanisms of eNOS-uncoupling seem multiple and complex. Recent research provides emerging evidence supporting an essential role of increased activity of arginases including arginase-I and arginase-II in causing eNOS-uncoupling, which results in vascular oxidative stress and inflammatory responses, and ultimately leading to vascular diseases. This review article will summarize the most recent findings on the functional roles of arginases in vascular diseases and/or dysfunctions and the underlying mechanisms in relation to oxidative stress and inflammations. Moreover, regulatory mechanisms of arginases in the vasculature are reviewed and the future perspectives of targeting arginases as therapeutic options in vascular diseases are discussed.

Oxidative stress and inflammation in the vascular wall are essential mechanisms of atherosclerosis and vascular dysfunctions associated with risk factors such as metabolic diseases, aging, hypertension, etc. Evidence has been provided that activation of the vascular endothelial cells in the presence of the risk factors promotes oxidative stress and vascular inflammatory responses, leading to acceleration of atherosclerotic vascular disease. Increasing number of studies from recent years demonstrates that uncoupling of endothelial nitric oxide synthase (eNOS), whereby the enzyme eNOS produces detrimental amount of superoxide anion <math id="M14"><msubsup><mrow><mtext>O</mtext></mrow><mrow><mn>2</mn></mrow><mrow><mo class="MathClass-bin">−</mo></mrow></msubsup></math> instead the vasoprotective nitric oxide (NO⋅), plays a critical role in vascular dysfunction under various pathophysiological conditions and in aging. The mechanisms of eNOS-uncoupling seem multiple and complex. Recent research provides emerging evidence supporting an essential role of increased activity of arginases including arginase-I and arginase-II in causing eNOS-uncoupling, which results in vascular oxidative stress and inflammatory responses, and ultimately leading to vascular diseases. This review article will summarize the most recent findings on the functional roles of arginases in vascular diseases and/or dysfunctions and the underlying mechanisms in relation to oxidative stress and inflammations. Moreover, regulatory mechanisms of arginases in the vasculature are reviewed and the future perspectives of targeting arginases as therapeutic options in vascular diseases are discussed.

Arginase: The Emerging Therapeutic Target for Vascular Oxidative Stress and Inflammation

Endothelial dysfunction is a characteristic feature in diabetes that contributes to the development of vascular disease. Recently, arginase has been implicated in triggering endothelial dysfunction in diabetic patients and animals by competing with endothelial nitric oxide synthase for substrate l</sc>-arginine. While most studies have focused on the coronary circulation and large conduit blood vessels, the role of arginase in mediating diabetic endothelial dysfunction in other vascular beds has not been fully investigated. In the present study, we determined whether arginase contributes to endothelial dysfunction in skeletal muscle arterioles of diabetic rats. Diabetes was induced in male Sprague Dawley rats by streptozotocin injection. Four weeks after streptozotocin administration, blood glucose, glycated hemoglobin, and vascular arginase activity were significantly increased. In addition, a significant increase in arginase I and II mRNA expression was detected in gracilis muscle arterioles of diabetic rats compared to age-matched, vehicle control animals. To examine endothelial function, first-order gracilis muscle arterioles were isolated, cannulated in a pressure myograph system, exposed to graded levels of luminal flow, and internal vessel diameter measured. Increases in luminal flow (0–50 μL/min) caused progressive vasodilation in arterioles isolated from control, normoglycemic animals. However, flow-induced vasodilation was absent in arterioles obtained from streptozotocin-treated rats. Acute in vitro pretreatment of blood vessels with the arginase inhibitors Nω-hydroxy-nor-l</sc>-arginine or S-(2-boronoethyl)-l</sc>-cysteine restored flow-induced responses in arterioles from diabetic rats and abolished differences between diabetic and control animals. Similarly, acute in vitro pretreatment with l</sc>-arginine returned flow-mediated vasodilation in vessels from diabetic animals to that of control rats. In contrast, d</sc>-arginine failed to restore flow-induced dilation in arterioles isolated from diabetic animals. Administration of sodium nitroprusside resulted in a similar degree of dilation in arterioles isolated from control or diabetic rats. In conclusion, the present study identifies arginase as an essential mediator of skeletal muscle arteriolar endothelial dysfunction in diabetes. The ability of arginase to induce endothelial dysfunction in skeletal muscle arterioles may further compromise glucose utilization and facilitate the development of hypertension in diabetes.

Endothelial dysfunction is a characteristic feature in diabetes that contributes to the development of vascular disease. Recently, arginase has been implicated in triggering endothelial dysfunction in diabetic patients and animals by competing with endothelial nitric oxide synthase for substrate L-arginine. While most studies have focused on the coronary circulation and large conduit blood vessels, the role of arginase in mediating diabetic endothelial dysfunction in other vascular beds has not been fully investigated. In the present study, we determined whether arginase contributes to endothelial dysfunction in skeletal muscle arterioles of diabetic rats. Diabetes was induced in male Sprague Dawley rats by streptozotocin injection. Four weeks after streptozotocin administration, blood glucose, glycated hemoglobin, and vascular arginase activity were significantly increased. In addition, a significant increase in arginase I and II mRNA expression was detected in gracilis muscle arterioles of diabetic rats compared to age-matched, vehicle control animals. To examine endothelial function, first-order gracilis muscle arterioles were isolated, cannulated in a pressure myograph system, exposed to graded levels of luminal flow, and internal vessel diameter measured. Increases in luminal flow (0–50 μL/min) caused progressive vasodilation in arterioles isolated from control, normoglycemic animals. However, flow-induced vasodilation was absent in arterioles obtained from streptozotocin-treated rats. Acute in vitro pretreatment of blood vessels with the arginase inhibitors Nω-hydroxy-nor-L-arginine or S-(2-boronoethyl)-L-cysteine restored flow-induced responses in arterioles from diabetic rats and abolished differences between diabetic and control animals. Similarly, acute in vitro pretreatment with L-arginine returned flow-mediated vasodilation in vessels from diabetic animals to that of control rats. In contrast, D-arginine failed to restore flow-induced dilation in arterioles isolated from diabetic animals. Administration of sodium nitroprusside resulted in a similar degree of dilation in arterioles isolated from control or diabetic rats. In conclusion, the present study identifies arginase as an essential mediator of skeletal muscle arteriolar endothelial dysfunction in diabetes. The ability of arginase to induce endothelial dysfunction in skeletal muscle arterioles may further compromise glucose utilization and facilitate the development of hypertension in diabetes.

Arginase Promotes Skeletal Muscle Arteriolar Endothelial Dysfunction in Diabetic Rats

Arginase metabolizes the semi-essential amino acid l</sc>-arginine to l</sc>-ornithine and urea. There are two distinct isoforms of arginase, arginase I and II, which are encoded by separate genes and display differences in tissue distribution, subcellular localization, and molecular regulation. Blood vessels express both arginase I and II but their distribution appears to be cell-, vessel-, and species-specific. Both isoforms of arginase are induced by numerous pathologic stimuli and contribute to vascular cell dysfunction and vessel wall remodeling in several diseases. Clinical and experimental studies have documented increases in the expression and/or activity of arginase I or II in blood vessels following arterial injury and in pulmonary and arterial hypertension, aging, and atherosclerosis. Significantly, pharmacological inhibition or genetic ablation of arginase in animals ameliorates abnormalities in vascular cells and normalizes blood vessel architecture and function in all of these pathological states. The detrimental effect of arginase in vascular remodeling is attributable to its ability to stimulate vascular smooth muscle cell and endothelial cell proliferation, and collagen deposition by promoting the synthesis of polyamines and l</sc>-proline, respectively. In addition, arginase adversely impacts arterial remodeling by directing macrophages toward an inflammatory phenotype. Moreover, the proliferative, fibrotic, and inflammatory actions of arginase in the vasculature are further amplified by its capacity to inhibit nitric oxide (NO) synthesis by competing with NO synthase for substrate, l</sc>-arginine. Pharmacologic or molecular approaches targeting specific isoforms of arginase represent a promising strategy in treating obstructive fibroproliferative vascular disease.

Arginase metabolizes the semi-essential amino acid L-arginine to L-ornithine and urea. There are two distinct isoforms of arginase, arginase I and II, which are encoded by separate genes and display differences in tissue distribution, subcellular localization, and molecular regulation. Blood vessels express both arginase I and II but their distribution appears to be cell-, vessel-, and species-specific. Both isoforms of arginase are induced by numerous pathologic stimuli and contribute to vascular cell dysfunction and vessel wall remodeling in several diseases. Clinical and experimental studies have documented increases in the expression and/or activity of arginase I or II in blood vessels following arterial injury and in pulmonary and arterial hypertension, aging, and atherosclerosis. Significantly, pharmacological inhibition or genetic ablation of arginase in animals ameliorates abnormalities in vascular cells and normalizes blood vessel architecture and function in all of these pathological states. The detrimental effect of arginase in vascular remodeling is attributable to its ability to stimulate vascular smooth muscle cell and endothelial cell proliferation, and collagen deposition by promoting the synthesis of polyamines and L-proline, respectively. In addition, arginase adversely impacts arterial remodeling by directing macrophages toward an inflammatory phenotype. Moreover, the proliferative, fibrotic, and inflammatory actions of arginase in the vasculature are further amplified by its capacity to inhibit nitric oxide (NO) synthesis by competing with NO synthase for substrate, L-arginine. Pharmacologic or molecular approaches targeting specific isoforms of arginase represent a promising strategy in treating obstructive fibroproliferative vascular disease.