Dual Role of Hydrogen Peroxide as an Oxidant in Pneumococcal Pneumonia.

Significance:Streptococcus pneumoniae (Spn), a facultative anaerobic Gram-positive human pathogen with increasing rates of penicillin and macrolide resistance, is a major cause of lower respiratory tract infections worldwide. Pneumococci are a primary agent of severe pneumonia in children younger than 5 years and of community-acquired pneumonia in adults. A major defense mechanism toward Spn is the generation of reactive oxygen species, including hydrogen peroxide (H2O2), during the oxidative burst of neutrophils and macrophages. Paradoxically, Spn produces high endogenous levels of H2O2 as a strategy to promote colonization. Recent Advances: Pneumococci, which express neither catalase nor common regulators of peroxide stress resistance, have developed unique mechanisms to protect themselves from H2O2. Spn generates high levels of H2O2 as a strategy to promote colonization. Production of H2O2 moreover constitutes an important virulence phenotype and its cellular activities overlap and complement those of other virulence factors, such as pneumolysin, in modulating host immune responses and promoting organ injury. Critical Issues: This review examines the dual role of H2O2 in pneumococcal pneumonia, from the viewpoint of both the pathogen (defense mechanisms, lytic activity toward competing pathogens, and virulence) and the resulting host-response (inflammasome activation, endoplasmic reticulum stress, and damage to the alveolar-capillary barrier in the lungs). Future Directions: An understanding of the complexity of H2O2-mediated host-pathogen interactions is necessary to develop novel strategies that target these processes to enhance lung function during severe pneumonia.

Age-Dependent Oxidative Stress Elevates Arginase 1 and Uncoupled Nitric Oxide Synthesis in Skeletal Muscle of Aged Mice.

Aging is associated with reduced muscle mass (sarcopenia) and poor bone quality (osteoporosis), which together increase the incidence of falls and bone fractures. It is widely appreciated that aging triggers systemic oxidative stress, which can impair myoblast cell survival and differentiation. We previously reported that arginase plays an important role in oxidative stress-dependent bone loss. We hypothesized that arginase activity is dysregulated with aging in muscles and may be involved in muscle pathophysiology. To investigate this, we analyzed arginase activity and its expression in skeletal muscles of young and aged mice. We found that arginase activity and arginase 1 expression were significantly elevated in aged muscles. We also demonstrated that SOD2, GPx1, and NOX2 increased with age in skeletal muscle. Most importantly, we also demonstrated elevated levels of peroxynitrite formation and uncoupling of eNOS in aged muscles. Our in vitro studies using C2C12 myoblasts showed that the oxidative stress treatment increased arginase activity, decreased cell survival, and increased apoptotic markers. These effects were reversed by treatment with an arginase inhibitor, 2(S)-amino-6-boronohexanoic acid (ABH). Our study provides strong evidence that L-arginine metabolism is altered in aged muscle and that arginase inhibition could be used as a novel therapeutic target for age-related muscle complications.

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.

Hyperglycemia-impaired aortic vasorelaxation mediated through arginase elevation: Role of stress kinase pathways.

Diabetes-induced vascular endothelial dysfunction has been reported to involve hyperglycemia-induced increases in arginase activity. However, upstream mediators of this effect are not clear. Here, we have tested involvement of Rho kinase, ERK1/2 and p38 MAPK pathways in this process. Studies were performed with aortas isolated from wild type or hemizygous arginase 1 knockout (Arg1+/-) mice and bovine aortic endothelial cells exposed to high glucose (HG, 25 mmol/l) or normal glucose (NG, 5.5 mmol/l) conditions for different times. Effects of inhibitors of arginase, p38 MAPK, ERK1/2 or ROCK and ex vivo adenoviral delivery of active Arg1 and inactive (D128-Arg1) cDNA were also determined. Exposure in wild type aorta or endothelial cells to HG significantly increased arginase activity and Arg1 expression and impaired aortic relaxation. Transduction of wild type aorta with active Arg1 cDNA impaired vascular relaxation, whereas inactive Arg1 had no effect. The HG-induced vascular endothelial dysfunction was associated with increased phosphorylation (activation) of ERK1/2 and p38 MAPK. Pretreatment with inhibitors of ERK1/2, p38 MAPK, ROCK or arginase blocked HG-induced elevation of arginase activity and Arg1 expression and prevented the vascular dysfunction. Inhibition of ROCK blunted the HG-induced activation of ERK1/2 and p38 MAPK. In summary, activated ROCK and subsequent activation of ERK1/2 or p38 MAPK elevates arginase activity and Arg1 expression in hyperglycemic states. Targeting this pathway may provide an effective means for preventing diabetes/hyperglycemia-induced vascular endothelial dysfunction.

Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses.

The lack of effective therapies to limit neurovascular injury in ischemic retinopathy is a major clinical problem. This study aimed to examine the role of ureohydrolase enzyme, arginase 1 (A1), in retinal ischemia-reperfusion (IR) injury. A1 competes with nitric oxide synthase (NOS) for their common substrate L-arginine. A1-mediated L-arginine depletion reduces nitric oxide (NO) formation by NOS leading to vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved. Studies were performed using wild-type (WT) mice, global A1+/- knockout (KO), endothelial-specific A1 KO, and myeloid-specific A1 KO mice subjected to retinal IR injury. Global as well as myeloid-specific A1 KO mice showed worsened IR-induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect, while treatment with PEGylated (PEG) A1 improved neuronal survival in WT mice. In addition, A1+/- KO mice showed worsened vascular injury manifested by increased acellular capillaries. Western blotting analysis of retinal tissue showed increased inflammatory and necroptotic markers with A1 deletion. In vitro experiments showed that macrophages lacking A1 exhibit increased inflammatory response upon LPS stimulation. PEG-A1 treatment dampened this inflammatory response and decreased the LPS-induced metabolic reprogramming. Moreover, intravitreal injection of A1 KO macrophages or systemic macrophage depletion with clodronate liposomes increased neuronal loss after IR injury. These results demonstrate that A1 reduces IR injury-induced retinal neurovascular degeneration via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage-mediated repair.

Arginase: A Multifaceted Enzyme Important in Health and Disease.

The arginase enzyme developed in early life forms and was maintained during evolution. As the last step in the urea cycle, arginase cleaves l-arginine to form urea and l-ornithine. The urea cycle provides protection against excess ammonia, while l-ornithine is needed for cell proliferation, collagen formation, and other physiological functions. In mammals, increases in arginase activity have been linked to dysfunction and pathologies of the cardiovascular system, kidney, and central nervous system and also to dysfunction of the immune system and cancer. Two important aspects of the excessive activity of arginase may be involved in diseases. First, overly active arginase can reduce the supply of l-arginine needed for the production of nitric oxide (NO) by NO synthase. Second, too much l-ornithine can lead to structural problems in the vasculature, neuronal toxicity, and abnormal growth of tumor cells. Seminal studies have demonstrated that increased formation of reactive oxygen species and key inflammatory mediators promote this pathological elevation of arginase activity. Here, we review the involvement of arginase in diseases affecting the cardiovascular, renal, and central nervous system and cancer and discuss the value of therapies targeting the elevated activity of arginase.

Netrin-1 is a novel regulator of vascular endothelial function in diabetes.

BACKGROUND: Netrin-1, a secreted laminin-like protein identified as an axon guidance molecule, has been shown to be of critical importance in the cardiovascular system. Recent studies have revealed pro-angiogenic, anti-apoptotic and anti-inflammatory properties of netrin-1 as well as cardioprotective actions against myocardial injury in diabetic mice. AIM: To examine the role of netrin-1 in diabetes-and high glucose (HG)-induced vascular endothelial dysfunction (VED) using netrin-1 transgenic mice (Tg3) and cultured bovine aortic endothelial cells (BAEC). MAIN OUTCOME: Overexpression of netrin-1 prevented diabetes-induced VED in aorta from diabetic mice and netrin-1 treatment attenuated HG-induced impairment of nitric oxide synthase (NOS) function in BAECs. METHODS AND RESULTS: Experiments were performed in Tg3 and littermate control (WT) mice rendered diabetic with streptozotocin (STZ) and in BAECs treated with HG (25 mmol/L). Levels of netrin-1 and its receptor DCC, markers of inflammation and apoptosis and vascular function were assessed in aortas from diabetic and non-diabetic Tg3 and WT mice. Vascular netrin-1 in WT mice was reduced under diabetic conditions. Aortas from non-diabetic Tg3 and WT mice showed similar maximum endothelium-dependent relaxation (MEDR) (83% and 87%, respectively). MEDR was markedly impaired in aorta from diabetic WT mice (51%). This effect was significantly blunted in Tg3 diabetic aortas (70%). Improved vascular relaxation in Tg3 diabetic mice was associated with increased levels of phospho-ERK1/2 and reduced levels of oxidant stress, NFκB, COX-2, p16INK4A, cleaved caspase-3 and p16 and p53 mRNA. Netrin-1 treatment prevented the HG-induced decrease in NO production and elevation of oxidative stress and apoptosis in BAECs. CONCLUSIONS: Diabetes decreases aortic levels of netrin-1. However, overexpression of netrin-1 attenuates diabetes-induced VED and limits the reduction of NO levels, while increasing expression of p-ERK1/2, and suppressing oxidative stress and inflammatory and apoptotic processes. Enhancement of netrin-1 function may be a useful therapeutic means for preventing vascular dysfunction in diabetes.

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.

Regulation of endothelial intracellular adenosine via adenosine kinase epigenetically modulates vascular inflammation.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Toll-like receptor 4 (TLR4) impairs nitric oxide contributing to Angiotensin II-induced cavernosal dysfunction.

AIM: Angiotensin II (AngII), a corpus cavernosum (CC) constrictor peptide, modulates Toll like receptor (TLR) expression, a key element of the innate immune system, contributing to impaired vascular function in pathological conditions. However, it is unknown whether TLR4 is involved in AngII-induced erectile dysfunction. In this study, we investigated whether TLR4 plays a role in cavernosal dysfunction caused by AngII upregulation. MATERIAL AND METHODS: Cavernosal smooth muscle cells (CSMC) from C57/BL6 mice were treated with AngII (0.1µM) or bacterial LPS (50ng/ml) for 12-24h and TLR4 expression was assessed. Mice were infused with AngII (90ng/min, 28days) and treated with anti-TLR4 antibody (0.1mg/daily, i.p.) for the last 14days of the treatment. CC tissue was used for functional studies and for Western blotting. Nitric Oxide Synthase (NOS) activity was measured by conversion of [3H]-l-arginine to [3H]-l-citrulline, systemic TNF-α levels by ELISA, and reactive oxygen species (ROS) by immunofluorescence. KEY FINDINGS: We report upregulation of TLR4 in CSMC following AngII or LPS stimulation. In AngII-infused mice, chronic treatment with anti-TLR4 antibody (28±2.1%) attenuates adrenergic CC contraction, which also ameliorates nitrergic (68.90±0.21 vs. 51.07±0.63, 8Hz, AngII-infused mice treated vs. non-treated). Decreased endothelial NOS expression, reduced NOS activity, and augmented levels of TNF-α, and ROS were found following AngII-infusion. These alterations were prevented, or at least decreased by anti-TLR4 antibody treatment. SIGNIFICANCE: Inhibition of TLR4 ameliorates AngII-impaired cavernosal relaxation, decreases TNF-α levels, and restores NO bioavailability, demonstrating that TLR4 partly mediates AngII-induced cavernosal dysfunction.

Obesity-induced vascular inflammation involves elevated arginase activity.

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 µM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

NOX2-Induced Activation of Arginase and Diabetes-Induced Retinal Endothelial Cell Senescence.

Increases in reactive oxygen species (ROS) and decreases in nitric oxide (NO) have been linked to vascular dysfunction during diabetic retinopathy (DR). Diabetes can reduce NO by increasing ROS and by increasing activity of arginase, which competes with nitric oxide synthase (NOS) for their commons substrate l-arginine. Increased ROS and decreased NO can cause premature endothelial cell (EC) senescence leading to defective vascular repair. We have previously demonstrated the involvement of NADPH oxidase 2 (NOX2)-derived ROS, decreased NO and overactive arginase in DR. Here, we investigated their impact on diabetes-induced EC senescence. Studies using diabetic mice and retinal ECs treated with high glucose or H2O2 showed that increases in ROS formation, elevated arginase expression and activity, and decreased NO formation led to premature EC senescence. NOX2 blockade or arginase inhibition prevented these effects. EC senescence was also increased by inhibition of NOS activity and this was prevented by treatment with a NO donor. These results indicate that diabetes/high glucose-induced activation of arginase and decreases in NO bioavailability accelerate EC senescence. NOX2-generated ROS contribute importantly to this process. Blockade of NOX2 or arginase represents a strategy to prevent diabetes-induced premature EC senescence by preserving NO bioavailability.

Mitochondrial targeting by dichloroacetate improves outcome following hemorrhagic shock.

Hemorrhagic shock is a leading cause of death in people under the age of 45 and accounts for almost half of trauma-related deaths. In order to develop a treatment strategy based on potentiating mitochondrial function, we investigated the effect of the orphan drug dichloroacetate (DCA) on survival in an animal model of hemorrhagic shock in the absence of fluid resuscitation. Hemorrhagic shock was induced in rats by withdrawing 60% of the blood volume and maintaining a hypotensive state. The studies demonstrated prolonged survival of rats subjected to hemorrhagic injury (HI) when treated with DCA. In separate experiments, using a fluid resuscitation model we studied mitochondrial functional alterations and changes in metabolic networks connected to mitochondria following HI and treatment with DCA. DCA treatment restored cardiac mitochondrial membrane potential and tissue ATP in the rats following HI. Treatment with DCA resulted in normalization of several metabolic and molecular parameters including plasma lactate and p-AMPK/AMPK, as well as Ach-mediated vascular relaxation. In conclusion we demonstrate that DCA can be successfully used in the treatment of hemorrhagic shock in the absence of fluid resuscitation; therefore DCA may be a good candidate in prolonged field care following severe blood loss.

Impaired Corpus Cavernosum Relaxation Is Accompanied by Increased Oxidative Stress and Up-Regulation of the Rho-Kinase Pathway in Diabetic (Db/Db) Mice.

Basal release of nitric oxide from endothelial cells modulates contractile activity in the corpus cavernosum via inhibition of the RhoA/Rho-kinase signaling pathway. We aimed to investigate nitric oxide bioavailability, oxidative stress and the Rho-kinase pathway in the relaxation of the corpus cavernosum of an obese and diabetic model of mice (db/db mice). We hypothesized that in db/db mice impaired relaxation induced by Rho-kinase inhibitor is accompanied by diminished NO bioavailability, increased oxidative stress and upregulation of the RhoA/Rho-kinase signalling pathway. Cavernosal strips from male lean and non-diabetic db/+ and db/db mice were mounted in myographs and isometric force in response to Rho-kinase inhibitor Y-27632 was recorded. Enzyme activity and protein expression of oxidative stress markers and key molecules of the RhoA/Rho-kinase pathway were analyzed. The Rho-kinase inhibitor Y-27632 concentration-dependently caused corpus cavernosum relaxation and inhibited cavernosal contractions. Nonetheless, a rightward shift in the curves obtained in corpus cavernosum of db/db mice was observed. Compared to db/+, this strain presented increased active RhoA, higher MYPT-1 phosphorylation stimulated by phenylephrine, and increased expression of ROKα and Rho-GEFs. Further, we observed normal expression of endothelial and neuronal NOS in corpus cavernosum of db/db mice. However, nitrate/nitrate (NOx) levels were diminished, suggesting decreased NO bioavailability. We measured the oxidant status and observed increased lipid peroxidation, with decreased SOD activity and expression. In conclusion, our data demonstrate that in db/db mice, upregulation of the RhoA/Rho-kinase signalling pathway was accompanied by decreased NO bioavailability and increased oxidative stress contributing to impaired relaxation of the corpus cavermosum of db/db mice.

Deregulation of arginase induces bone complications in high-fat/high-sucrose diet diabetic mouse model.

A balanced diet is crucial for healthy development and prevention of musculoskeletal related diseases. Diets high in fat content are known to cause obesity, diabetes and a number of other disease states. Our group and others have previously reported that activity of the urea cycle enzyme arginase is involved in diabetes-induced dysregulation of vascular function due to decreases in nitric oxide formation. We hypothesized that diabetes may also elevate arginase activity in bone and bone marrow, which could lead to bone-related complications. To test this we determined the effects of diabetes on expression and activity of arginase, in bone and bone marrow stromal cells (BMSCs). We demonstrated that arginase 1 is abundantly present in the bone and BMSCs. We also demonstrated that arginase activity and expression in bone and bone marrow is up-regulated in models of diabetes induced by HFHS diet and streptozotocin (STZ). HFHS diet down-regulated expression of healthy bone metabolism markers (BMP2, COL-1, ALP, and RUNX2) and reduced bone mineral density, bone volume and trabecular thickness. However, treatment with an arginase inhibitor (ABH) prevented these bone-related complications of diabetes. In-vitro study of BMSCs showed that high glucose treatment increased arginase activity and decreased nitric oxide production. These effects were reversed by treatment with an arginase inhibitor (ABH). Our study provides evidence that deregulation of l-arginine metabolism plays a vital role in HFHS diet-induced diabetic complications and that these complications can be prevented by treatment with arginase inhibitors. The modulation of l-arginine metabolism in disease could offer a novel therapeutic approach for osteoporosis and other musculoskeletal related diseases.

Arginase: an old enzyme with new tricks.

Arginase has roots in early life-forms. It converts L-arginine to urea and ornithine. The former provides protection against NH3; the latter serves to stimulate cell growth and other physiological functions. Excessive arginase activity in mammals has been associated with cardiovascular and nervous system dysfunction and disease. Two relevant aspects of this elevated activity may be involved in these disease states. First, excessive arginase activity reduces the supply of L-arginine needed by nitric oxide (NO) synthase to produce NO. Second, excessive production of ornithine leads to vascular structural problems and neural toxicity. Recent research has identified inflammatory agents and reactive oxygen species (ROS) as drivers of this pathologic elevation of arginase activity and expression. We review the involvement of arginase in cardiovascular and nervous system dysfunction, and discuss potential therapeutic interventions targeting excess arginase.

Angiotensin II-induced arterial thickening, fibrosis and stiffening involves elevated arginase function.

BACKGROUND: Arterial stiffness (AS) is an independent risk factor for cardiovascular morbidity/mortality. Smooth muscle cell (SMC) proliferation and increased collagen synthesis are key features in development of AS. Arginase (ARG), an enzyme implicated in many cardiovascular diseases, can compete with nitric oxide (NO) synthase for their common substrate, L-arginine. Increased arginase can also provide ornithine for synthesis of polyamines via ornithine decarboxylase (ODC) and proline/collagen via ornithine aminotransferase (OAT), leading to vascular cell proliferation and collagen formation, respectively. We hypothesized that elevated arginase activity is involved in Ang II-induced arterial thickening, fibrosis, and stiffness and that limiting its activity can prevent these changes. METHODS AND RESULTS: We tested this by studies in mice lacking one copy of the ARG1 gene that were treated with angiotensin II (Ang II, 4 weeks). Studies were also performed in rat aortic Ang II-treated SMC. In WT mice treated with Ang II, we observed aortic stiffening (pulse wave velocity) and aortic and coronary fibrosis and thickening that were associated with increases in ARG1 and ODC expression/activity, proliferating cell nuclear antigen, hydroxyproline levels, and collagen 1 protein expression. ARG1 deletion prevented each of these alterations. Furthermore, exposure of SMC to Ang II (1 µM, 48 hrs) increased ARG1 expression, ARG activity, ODC mRNA and activity, cell proliferation, collagen 1 protein expression and hydroxyproline content. Treatment with ABH prevented these changes. CONCLUSION: Arginase 1 is crucially involved in Ang II-induced SMC proliferation and arterial fibrosis and stiffness and represents a promising therapeutic target.