Arginase Inhibitor Research Articles

Arginase inhibition prevents the development of hypertension and improves insulin resistance in obese rats.

This study investigated the temporal activation of arginase in obese Zucker rats (ZR) and determined if arginase inhibition prevents the development of hypertension and improves insulin resistance in these animals. Arginase activity, plasma arginine and nitric oxide (NO) concentration, blood pressure, and insulin resistance were measured in lean and obese animals. There was a chronological increase in vascular and plasma arginase activity in obese ZR beginning at 8weeks of age. The increase in arginase activity in obese animals was associated with a decrease in insulin sensitivity and circulating levels of arginine and NO. The rise in arginase activity also preceded the increase in blood pressure in obese ZR detected at 12weeks of age. Chronic treatment of 8-week-old obese animals with an arginase inhibitor or L-arginine for 4weeks prevented the development of hypertension and improved plasma concentrations of arginine and NO. Arginase inhibition also improved insulin sensitivity in obese ZR while L-arginine supplementation had no effect. In conclusion, arginase inhibition prevents the development of hypertension and improves insulin sensitivity while L-arginine administration only mitigates hypertension in obese animals. Arginase represents a promising therapeutic target in ameliorating obesity-associated vascular and metabolic dysfunction.

990 Targeting metabolism reprogramming of keratinocytes improves psoriasis-like skin inflammation

Keratinocyte-intrinsic metabolic reprogramming is the major fuel to epidermal hyperplasia in psoriasis, yet the underlying cause is poorly understood. We report that in psoriatic lesions, Protein Phosphatase 6 (PP6) is diminished in the epidermis, and its levels negatively correlate with the disease severity. Mice with genetic deficiency of Pp6 in keratinocytes spontaneously develop psoriasis-like skin phenotype resembling psoriasis clinically, histologically, in its gene expression profile and in its response to therapy. Mechanistically, Pp6-/- keratinocytes rely on inordinate ornithine cycle and enhanced oxidative phosphorylation (OXPHOS) to hyper-proliferate, mediated by increased Arginase-1 (Arg1) production resulting from the activation of CCAAT/enhancer-binding protein beta (C/EBPβ) and elevated levels of mitochondrial respiration chain components. Treating imiquimod-induced mouse model of psoriasis with an arginase inhibitor or (and) an OXPHOS inhibitor markedly improves the skin inflammation. Thus, our data reveal for the first time the molecular basis and the functional significance of keratinocyte metabolic adaptations in psoriasis, bringing forth novel therapeutic strategies targeting keratinocyte metabolism to treat psoriasis.

Characterization of the Electrochemical Detection of NG-Hydroxy-L-Arginine

NG-Hydroxy-L-arginine (NOHA) is a suspected biomarker in breast cancer due to its involvement in the L-arginine metabolism [1]. Previous research in human breast cancer cells has shown that NOHA, a potent arginase inhibitor, has an antiproliferative and apoptotic actions on the arginase-expressing cells [1]. NOHA is a stable intermediate product in the production of NO from the consumption of L-arginine [1]. L-ornithine and L-citrulline are also involved in the metabolism of L-arginine through the urea cycle; the urea cycle has been known to be disrupted in cancer patients [1]. Early detection of cancer is important for rapid treatment options to be available [2]. Electrochemical detection is an established, cost-effective method that has potential to detect the onset of cancer because of its ability to successfully detect low levels of analyte concentrations. Before a robust biosensor sensor can be made the characterization of the electrochemical detection of NOHA needs to be done. Electrochemical activity of NOHA was investigated with cyclic voltammetry and differential pulse voltammetry. NOHA was found to have an oxidation and reduction peak at 355 mV and 188 mV vs. Ag/AgCl respectively. In cyclic voltammetric measurements a scan rate of 50 mVs-1 was used over the applied potential of -0.2 to 0.6 V vs Ag/AgCl. Triplicate data was obtained for serial dilutions of NOHA in two different buffers; Phosphate Buffer Saline solution pH 7.3 (PBS) and 100uM L-Ornithine, 10uM L-Citrulline, and 100uM L-Arginine in PBS (LOCA). The electrochemical detection of NOHA in a LOCA solution was done to understand the effect of a urea cycle metabolite mixture on electrode fowling and sensitivity. A shift in oxidation peak voltage was found for the LOCA solution by 5 mV to 360 mV. The sensitivity of the NOHA in PBS and LOCA solutions were found to be 5.4 nA/uM and 4.8 nA/uM respectively. In addition to our multiple data points to discuss sensitivity and reproducibility in the detection of NOHA, we will report kinetic information including the number of electrons transferred and diffusivity. We will also discuss our progress to decrease the limit-of-detection of NOHA in an effort to make a more sensitive bioanalyte sensor. [1] R. Singh, S. Pervin, A. Karimi, S. Cederbaum, and G. Chaudhuri, “Arginase activity in human breast cancer cell lines: Nω-hydroxy-L-arginine selectively inhibits cell proliferation and induces apoptosis in MDA-MB-468 cells,” Cancer Res., vol. 60, pp. 3305–3312, (2000) [2] Zhen Zhang, Robert C. Bast, Jr., Yinhua Yu, Jinong Li, Lori J. Sokoll, Alex J. Rai, Jason M. Rosenzweig, Bonnie Cameron, Young Y. Wang, Xiao-Ying Meng, Andrew Berchuck, Carolien van Haaften-Day, and Neville F. Hacker, Henk W. A. de Bruijn, Ate G. J. van der Zee, Ian J. Jacobs, Eric T. Fung, and D. W. Chan, “Three Biomarkers Identified from Serum Proteomic Analysis for the Detection of Early Stage Ovarian Cancer,” Cancer Res., vol. 64, pp. 5882–5890, (2004)

Mechanisms of Retinal Ischemia/Reperfusion Injury: Arginase and the Mitochondria

ObjectiveOur group has previously shown that the ureahydrolyase mitochondrial isoenzyme, arginase 2 (A2) is involved in ischemia reperfusion (I/R)‐induced retinal neurovascular degeneration. We found that A2 homozygous deletion significantly protected against necroptosis, neurovascular degeneration, retinal thinning, and impairment of retinal function. Interestingly, our group has also shown a link between hypoxia‐induced increases in A2 and impaired angiogenesis in endothelial cells. The cytosolic isoform arginase 1 (A1) was not affected which suggests distinct roles of A1 versus A2 under hypoxic conditions. However, the exact mechanisms of arginase‐induced neurovascular damage in retinal I/R are not yet known. In the current study, we investigated the involvement of arginase in retinal I/R injury in vivo and in vitro in relation to mitochondrial function.MethodsI/R insult was conducted on the right eyes of wild type (WT) and A2 homozygous knockout (A2−/−) mice. The left eyes were used as sham controls. Bovine retinal endothelial cells (BRECs) subjected to oxygen glucose deprivation (OGD/R) were used as an in vitro model. Cells were treated with the arginase inhibitor, Amino‐2‐Borono‐6‐Hexanoic Acid (ABH, 100μM), recombinant Pegylated A1 (PegA1, 1μg/ml), or vehicle. Western blot was used to evaluate different protein levels. Mitochondria structure and function were evaluated using Rhodamine 123 labeling and Seahorse XF analyzer respectively.ResultsWe found that the mitochondrial fission protein, dynamin related protein (Drp1) was significantly increased in WT I/R retinas compared to sham controls at 3 and 6 hours. With A2 deletion, the Drp1 levels were also elevated after I/R but were significantly reduced to sham control levels at 6 hours. In cell culture studies, A2 protein levels were significantly increased after 6h OGD/ 6h R. The cleavage of poly ADP‐ribose polymerase (PARP) and phosphorylation of p38‐MAPK were significantly increased after OGD/R. Arginase inhibition significantly reduced the phosphorylation of p38 and slightly reduced PARP cleavage. Western blot analysis showed increased Drp1 expression and mitochondria labeling showed more fragmented mitochondria after OGD/R. With arginase inhibition, the mitochondria were restored and resumed a more normal morphology with significantly less Drp1 expression. Seahorse XF analysis of the oxygen consumption rate (OCR) showed a significant decrease in maximal respiration and spare respiratory capacity after OGD/R indicating impaired mitochondrial function. Treatment with PegA1 significantly prevented the OGD/R‐induced mitochondrial dysfunction as reflected by increased spare respiratory capacity and maximal respiration. However, treatment with the non‐selective arginase inhibitor (ABH) did not improve mitochondrial function under OGD/R conditions.ConclusionsThis study is the first to show that A1 and A2 could be playing different roles under ischemic conditions by affecting the mitochondrial function in distinct ways. Furthermore, PegA1 therapy may offer a new strategy for limiting hypoxia‐induced impairment of angiogenic function.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Arginase inhibition improves vascular function and restores NO production in diabetic conditions

Cardiovascular complications of diabetes are a leading cause of morbidity and mortality. Vascular endothelial dysfunction (VED) is strongly implicated in the pathogenesis of diabetic vascular complications. Diabetes has been linked to elevated arginase and associated with vascular endothelial dysfunction. The pathological process is characterized by impaired endothelial cell production of nitric oxide (NO) and/or decreased NO bioavailability. NO is produced by activity of endothelial NO synthase (eNOS) on its substrate L‐arginine. Arginase in ECs also uses L‐arginine as a substrate to produce urea and ornithine. Thus, elevated levels of arginase will limit availability of L‐arginine to eNOS and decrease NO production.We performed analysis of arginase activity in type 2 diabetes mellitus (T2DM). Arginase activity was elevated in diabetes patients as compared to age‐matched healthy volunteers. Levels of arginase activity had a positive correlation with HbA1c levels in diabetic patients (R2=0.8 Pearson r=0.87). Cell studies also agreed with these findings, as high glucose (25 mmol/L, 72 hrs) treatment to ECs resulted in a significant increase in arginase activity. This increase in arginase activity was concomitant with a 27% drop in NO produced by EC. Inhibition of arginase by ABH (100 uM) restored NO level to normal.The effects of L‐citrulline, a natural arginase inhibitor, on inhibiting arginase activity in T2DM patients was assessed. Twenty five patients received L‐citrulline supplements (2000 mg/day) for 1 month. Arginase activity was decreased 30% in T2DM patients after taking L‐citrulline supplements. There was a modest improvement on HbA1c levels in these patients. The effect on L‐citrulline on arginase activity was also measured in BAECs grown in high glucose conditions. L‐citrulline 100μM completely prevented the increase in arginase activity and restored NO production levels. These data indicate that inhibiting arginase activity can have therapeutic benefits in diabetic patients by preventing vascular dysfunction and maintaining NO levels.Our results indicate that arginase can be regarded as a novel marker for the vascular complications of diabetes. Drugs targeting arginase or its signaling pathway may show benefits in delaying or preventing these vascular complications.Support or Funding InformationThe University of Jordan deanship of researchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effectiveness of arginase inhibitors against experimentally induced stroke.

Stroke is a lethal disease, but it disables more than it kills. Stroke is the second leading cause of death and the most frequent cause of permanent disability in adults worldwide, with 90% of survivors having residual deficits. The pathophysiology of stroke is complex and involves a strong inflammatory response associated with oxidative stress and activation of several proteolytic enzymes. The current study was designed to investigate the effect of arginase inhibitors (L-citruline and L-ornithine) against ischemic stroke induced in rats by middle cerebral artery occlusion (MCAO). MCAO resulted in alteration in rat behavior, brain infarct, and edema associated with disruption of the blood-brain barrier (BBB). This was mediated through overexpression of arginase I and II, inducible NOS (iNOS), malondialdehyde (MDA), advanced glycation end products (AGEs), TNF-α, and IL-1β and downregulation of endothelial nitric oxide synthase (eNOS). Treatment with L-citruline and L-ornithine and the standard neuroprotective drug cerebrolysin ameliorated all the deleterious effects of stroke. These results indicate the possible use of arginase inhibitors in the treatment of stroke after suitable clinical trials are done.

Arginase controls soluble vascular endothelial growth factor receptor 1 (sFlt1) to maintain pregnancy homeostasis

Excessive soluble fms-like tyrosine kinase-1 (sFlt-1) has been strongly implicated in preeclampsia. An increase in the serum sFlt-1 level occurs before the onset of preeclampsia, and the sFlt-1 level is already higher in women who are predisposed to preeclampsia than in normotensive pregnant women. This study aimed to investigate the relation between arginase and sFlt-1 in the plasma of preeclamptic women and normotensive pregnant women.We suggested that a regulatory mechanism exists that suppresses the level of sFlt-1. The relationship between arginase, one of the nitric oxide (NO) modulators, and sFlt-1 was examined. First, the pregnant women were divided into 4 groups: group 1, sFlt-1 <6000 pg/ml and arginase activity <110 U/L; group 2, sFlt-1 ≥6000 pg/ml and arginase activity <110 U/L; group 3, sFlt-1 ≥6000 pg/ml and arginase activity ≥110 U/L; and group 4, sFlt-1 <6000 pg/ml and arginase activity ≥110 U/L. Groups 2 and 3 comprised preeclamptic women. The preeclampsia/normotensive ratio increased from groups 1 to 3. Under the higher sFlt-1 condition, lower arginase activity was associated with lower occurrence of preeclampsia. Next, in human umbilical endothelial vein cells (HUVECs), a slightly higher concentration of sFlt-1, as in group 2, reduced arginase expression and arginase activity, and S-(2-boronoethyl)-l-cysteine (BEC; arginase inhibitor) impaired sFlt-1 secretion. In contrast, a higher level of sFlt-1 increased arginase expression and activity in HUVECs, as in group 3. These results showed that arginase controlled sFlt-1 elevation to some extent.In conclusion, our results suggest the existence of a mechanism to maintain the level of sFlt-1. Soluble Flt-1 negatively regulated itself against increasing serum sFlt-1 in preeclampsia. Moreover, this study revealed that arginase inhibitors are a potential treatment option for preeclampsia.

L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 μM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.

Homoarginine and inhibition of human arginase activity: kinetic characterization and biological relevance

The inhibition of arginase, resulting in higher arginine (ARG) availability for nitric oxide synthesis, may account for the putative protective effect of homoarginine (HOMOARG) against atherosclerosis and cardiovascular disease. However, uncertainty exists regarding the significance of HOMOARG-induced arginase inhibition in vivo. A novel UPLC-MS method, measuring the conversion of ARG to ornithine (ORN), was developed to determine arginase 1 and arginase 2 inhibition by HOMOARG, lysine (LYS), proline (PRO), agmatine (AG), asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and NG-Monomethyl-L-arginine (L-NMMA). Plasma HOMOARG, ARG and ORN concentrations were further measured in 50 healthy older adults >65 years (27 males and 23 females). HOMOARG inhibited arginase 1 with IC50 and Ki values of 8.14 ± 0.52 mM and 6.1 ± 0.50 mM, and arginase 2 with IC50 and Ki values of 2.52 ± 0.01 mM and 1.73 ± 0.10 mM, respectively. Both arginase isoforms retained 90% activity vs. control when physiological HOMOARG concentrations (1–10 µM) were used. In partial correlation analysis, plasma HOMOARG was not associated with ARG (P = 0.38) or ARG/ORN ratio (P = 0.73) in older adults. Our results suggest that arginase inhibition is unlikely to play a significant role in the reported cardio-protective effects of HOMOARG.

Bryophyllum pinnatum inhibits arginase II activity and prevents oxidative damage occasioned by carbon tetrachloride (CCl4) in rats

Bryophyllum pinnatum (B. pinnatum) (Lam.) Oken is used in tropical Africa for the treatment of several diseases such as kidney and urinary disorders. This study was aimed to evaluate the effect of B. pinnatum on arginase II activity and its prevention against renal oxidative damage occasioned by CCl4 in rats. Rats were randomly divided into six groups; group I served as the control, group II served as carbon tetrachloride (CCl4) intoxicated group, group III–V animals were pre-treated with silymarin (25 mg/kg body weight), 25 mg/kg body weight aqueous extracts of Bryophyllum pinnatum (AEBP) and 50 mg/kg body weight AEBP, respectively, for 14 days, followed by a single injection of CCl4. Group VI rats received AEBP only (50 mg/kg body weight). Results obtained revealed that CCl4 intoxication significantly increased (p < 0.05) the levels of renal markers (serum urea, creatinine and arginase II) in rats when compared to the control group. Further, oxidative stress status appeared in CCl4-intoxicated rats, as evidence by significant elevation in malondialdehyde (MDA), with concomitant decrease in levels of functional sulfhydryl groups (SH), antioxidant enzymes and nitric oxide in rats’ kidney. These adverse changes, due to CCl4 intoxication in rats, were however, prevented by pre-treatment with AEBP leaves (25 and 50 mg/kg body weight). The inhibition of arginase II, as well as increased antioxidant status by AEBP in CCl4-intoxicated rats suggests that B. pinnatum can protect kidney against CCl4-induced oxidative damage.

Cytokine Signaling Protein 3 Deficiency in Myeloid Cells Promotes Retinal Degeneration and Angiogenesis through Arginase-1 Up-Regulation in Experimental Autoimmune Uveoretinitis

The suppressor of cytokine signaling protein 3 (SOCS3) critically controls immune cell activation, although its role in macrophage polarization and function remains controversial. Using experimental autoimmune uveoretinitis (EAU) as a model, we show that inflammation-mediated retinal degeneration is exaggerated and retinal angiogenesis is accelerated in mice with SOCS3 deficiency in myeloid cells (LysMCre/+SOCS3fl/fl). At the acute stage of EAU, the population of infiltrating neutrophils was increased and the population of macrophages decreased in LysMCre/+SOCS3fl/fl mice compared with that in wild-type (WT) mice. Real-time RT-PCR showed that the expression of tumor necrosis factor-α, IL-1β, interferon-γ, granulocyte-macrophage colony-stimulating factor, and arginase-1 was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina in contrast to the WT EAU retina. The percentage of arginase-1+ infiltrating cells was significantly higher in the LysMCre/+SOCS3fl/fl EAU retina than that in the WT EAU retina. In addition, bone marrow-derived macrophages and neutrophils from the LysMCre/+SOCS3fl/fl mice express significantly higher levels of chemokine (C-C motif) ligand 2 and arginase-1 compared with those from WT mice. Inhibition of arginase using an l-arginine analog amino-2-borono-6-hexanoic suppressed inflammation-induced retinal angiogenesis without affecting the severity of inflammation. Our results suggest that SOCS3 critically controls the phenotype and function of macrophages and neutrophils under inflammatory conditions and loss of SOCS3 promotes the angiogenic phenotype of the cells through up-regulation of arginase-1.

Endoplasmic reticulum stress induced Lox-1+ CD15+ polymorphonuclear myeloid-derived suppressor cells in hepatocellular carcinoma and associated with poor prognsis.

38 Background: A recent study indicated that Lectin-type oxidized LDL receptor-1 (LOX-1) was a distinct surface marker for human polymorphonuclears myeloid-derived suppressor cells (PMN-MDSC). The present study was aimed to investigate the existence LOX-1 PMN-MDSC in hepatocellular carcinoma (HCC) patients, the latent mechanism and their association with clinical parameters. Methods: 30 HCC patients and 30 health control were included. LOX-1+CD15+ PMN-MDSCs were investigated. Results: LOX-1+CD15+ PMN-MDSC were significantly elevated in both WB and PBMC of HCC patients compared with healthy control. LOX-1+CD15+ PMN-MDSC were more abundant in PBMC than WB. Addition of PMN-MDSCs resulted in significantly reduced proliferation and IFN-γ production of T cells with a dosage dependent manner. LOX-1-CD15+ PMNs present no suppressive function. The suppression on T cell proliferation and IFN-γ production was reversed by ROS inhibitor and Arginase inhibitor. ROS level of LOX-1+CD15+ PMN by DCFDA were higher in LOX-1+CD15+ PMN-MDSCs than LOX-1-CD15+ PMNs, as well as the mRNA levels of the NADPH oxidase NOX2. Meanwhile, the expression of arginase I and activity of arginase were also significantly raised in LOX-1+CD15+ PMN-MDSCs. LOX-1+CD15+ PMN-MDSCs displayed significantly higher expression of spliced X-box–binding protein 1 (sXBP1), ATF3 and CCAAT/enhancer binding protein (CHOP) were higher. For HCC patients, LOX-1+CD15+ PMN-MDSCs in WB were positively related to Cancer of the Liver Italian Program (CLIP) score. Conclusions: LOX-1+CD15+ PMN-MDSC were elevated in HCC patients and suppressed T cell proliferation through ROS/Arg I pathway with ER stress as a potential feature. LOX-1+CD15+ PMN-MDSC presented positive association with the prognosis of HCC patients.

Gold Nanoparticle-Based Photoluminescent Nanoswitch Controlled by Host-Guest Recognition and Enzymatic Hydrolysis for Arginase Activity Assay.

The development of simple yet powerful methods for monitoring enzyme activity is of great significance. Herein, a facile, convenient, cost-effective, and continuous fluorescent method for the detection of arginase and its inhibitor has been reported based on a host-guest interaction-controlled and enzymatic hydrolysis-controlled luminescent nanoswitch. The fluorescence intensity of 6-aza-2-thiothymine-stabilized gold nanoparticle (ATT-AuNP) is enhanced by l-arginine, owing to the formation of a supramolecular host-guest assembly between the guanidine group of l-arginine and ATT molecules capped on the AuNP surface. However, hydrolysis of l-arginine, catalyzed by arginase, leads to a decrease in the fluorescence intensity of l-arginine/ATT-AuNPs hybrids. Upon incorporation of the arginase inhibitor l-norvaline, the fluorescence of the ATT-AuNP-based detecting system is restored. The linear range of arginase activity determination is from 0.0625 to 1.15 U/mL and the limit of detection is 0.056 U/mL. The half-maximal inhibition value IC50 of l-norvaline is determined to be 5.6 mM. The practicability of this luminescent nanoswitch is validated by assaying the arginase activity in rat liver and monitoring the response of rat liver arginase to pharmacological agent. Compared to the existing fluorescent method of arginase activity assay, the approach demonstrated here does not involve any complicated technical manipulation, thereby greatly simplifying the detection steps. We propose that this AuNP-based luminescent nanoswitch would find wide applications in the field of life sciences and medicine.

Inhibition of arginase via jugular infusion of Nω-hydroxy-nor-l-arginine inhibits casein synthesis in lactating dairy cows

A previous in vitro study revealed that Arg elicits positive effects on casein synthesis through alterations of the Arg-ornithine pathway in bovine mammary epithelial cells. The main purpose of this work was to determine the effects of arginase inhibition using Nω-hydroxy-nor-l-arginine (nor-NOHA) on milk protein synthesis in vivo. Six healthy Chinese Holstein cows with similar body weight (550.0 ± 20 kg; means ± standard deviation), parity (4), body condition score (3.0), milk yield (21.0 ± 1.0 kg), and days in milk (80 ± 2) were selected and randomly assigned to 3 treatments in a replicated 3 × 3 Latin square design with 22 d for each period (7 d for infusion and 15 d for washout). The treatments were (1) control: saline infusion; (2) nor-NOHA: infusion of 125 mg/L of nor-NOHA; (3) nor-NOHA + Arg: infusion of 125 mg/L of nor-NOHA with 9.42 g/L of Arg. The activity of enzymes related to Arg metabolism, milk protein synthesis, and expression of AA transporters was determined. The infusion of nor-NOHA decreased the activity of arginase but had no effect on the activity of ornithine decarboxylase and nitric oxide synthase in serum, and these responses were the same at the gene expression level in mammary gland. In addition, the infusion of nor-NOHA also reduced protein and fat synthesis in milk but had no effect on milk yield. When Arg was infused with nor-NOHA, the activity of total arginase, ornithine decarboxylase, and nitric oxide synthase, and the concentration of casein, protein, and fat in milk did not change compared with the nor-NOHA group, but the milk protein yield, the expression of some Arg transporters (SLC7A5 and SLC7A8), and milk yield increased. Overall, results verified previous in vitro findings indicating that synthesis of casein protein is closely regulated by the Arg-ornithine pathway in bovine mammary gland.

Attenuated cutaneous microvascular function in healthy young African Americans: Role of intradermal l-arginine supplementation

It has been established that endothelial function in conduit vessels is reduced in young African Americans (AA) relative to Caucasian Americans (CA). However, less is known regarding endothelial function in microvasculature of young AA. We hypothesized that microvascular function in response to local heating of skin is attenuated in young AA relative to age-matched CA due largely to the lack of NO bioavailability, which is in turn improved by intradermal l-arginine supplementation and/or inhibition of arginase. Nine AA and nine CA adults participated in this study. Participants were instrumented with four microdialysis membranes in the cutaneous vasculature of one forearm and were randomly assigned to receive 1) lactated Ringer's solution as a control site; 2) 20 mM NG-nitro-l-arginine (l-NAME) to inhibit NO synthase activity; 3) 10 mM l-arginine to local supplement l-arginine; or 4) a combination of 5.0 mM (S)-(2‑boronoethyl)-l-cysteine-HCL (BEC) and 5.0 mM Nω-hydroxy-nor-l-arginine (nor-NOHA) at a rate of 2.0 μl/min to locally inhibit arginase activity. Cutaneous vascular conductance (CVC) was calculated as red blood cell flux divided by mean arterial pressure. All CVC data were presented as a percentage of maximal CVC (%CVCmax) that was determined by maximal cutaneous vasodilation induced by 44 °C heating plus sodium nitroprusside administration. The response during the 42 °C local heating plateau was blunted in the AA at the control site (CA: 84 ± 12 vs. AA: 62 ± 6 vs. %CVCmax; P < 0.001). This response was improved in AA at the l-arginine site (Control: 62 ± 6 vs. l-arginine: 70 ± 18%CVCmax; P < 0.05) but not in the arginase inhibited site (Control: 62 ± 6 vs. Arginase inhibited: 62 ± 13%CVCmax; P = 0.91). In addition, the AA group had an attenuated NO contribution to the plateau phase during 42 °C local heating relative to the CA group (CA: 56 ± 14 vs. AA: 44 ± 6 Δ %CVCmax; P < 0.001). These findings suggest that 1) cutaneous microvascular function in response to local heating is blunted in young AA when compared to age-matched young CA; 2) this attenuated response is partly related to decrease in NO bioavailability in young AA; and 3) a local infusion of l-arginine, but not arginase inhibition, improves cutaneous microvascular responses to local heating in young AA relative to CA.

Arginase II inhibition prevents interleukin-8 production through regulation of p38 MAPK phosphorylation activated by loss of mitochondrial membrane potential in nLDL-stimulated hAoSMCs

Arginase inhibition exhibits beneficial effects in vascular endothelial and smooth muscle cells. In human aortic smooth muscle cells (hAoSMCs), native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. Therefore, we examined the effect of arginase inhibition on IL-8 production and the underlying mechanism. In hAoSMCs, reverse transcription–PCR, western blotting and immunocytochemistry with MitoTracker confirmed that arginase II was confined predominantly to mitochondria. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine ethyl ester. The MMP decreased upon nLDL stimulation but was restored upon arginase inhibition. MMP loss caused by nLDL was prevented by treatment with the intracellular Ca2+ chelator BAPTA-AM. In mitochondrial Ca2+ measurements using Rhod-2 AM, increased mitochondrial Ca2+ levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca2+ movement. The nLDL-induced MMP change resulted in p38 mitogen-activated protein kinase (MAPK) phosphorylation and IL-8 production and was prevented by the arginase inhibitors BAPTA and ruthenium 360. In isolated AoSMCs from ApoE−/− mice fed a high-cholesterol diet, arginase activity, p38 MAPK phosphorylation, spermine and mitochondrial Ca2+ levels and keratinocyte-derived chemokine (KC) production were increased compared with wild-type (WT) mice. However, in AoSMCs isolated from arginase II-null mice, increases in MMP and decreases in mitochondrial Ca2+ levels were noted compared with WT and were associated with p38 MAPK activation and IL-8 production. These data suggest that arginase activity regulates the change in MMP through Ca2+ uptake that is essential for p38 MAPK phosphorylation and IL-8 production.

Phenolic Compounds as Arginase Inhibitors: New Insights Regarding Endothelial Dysfunction Treatment.

Endothelial dysfunction is characterised by the low bioavailability of nitric oxide with a relevant negative impact on the nitric oxide/cGMP pathway. The loss of nitric oxide/cGMP signaling may be caused by an increased arginase activity. Plant-derived substances, especially polyphenols, are compounds that have the potential to inhibit arginase activity and they may represent an attractive therapeutic option to combat clinical outcomes related to endothelial dysfunction. An extensive review was carried out using all available data published in English in the Pubmed database, and without restriction regarding the year of publication. Despite the increased number of new substances that have been tested as arginase inhibitors, it is rare to find a compound that satisfies all the toxicological criteria to be used in the development of a new drug. On the other hand, recent data have shown that substances from plants have great potential to be applied as arginase inhibitors, most of which are polyphenols. Of the relevant mechanisms in this process, the inhibition of arginase by natural products seems to act against endothelial dysfunction by reestablishing the vascular function and elevating nitric oxide levels (by increasing the amounts of substrate (L-arginine, and endothelial nitric oxide synthase activation and stabilisation) as well as decreasing the generation of reactive species (formed by uncoupledendothelial nitric oxide synthase). This review summarises several topics regarding arginase inhibition by natural substances as well as indicating this pathway as an emergent strategy to elevate nitric oxide levels in disorders involving endothelial dysfunction. In addition, some aspects regarding structural activity and future perspectives are discussed.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

PurposeVascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms.Materials and MethodsIsolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin.ResultsLimonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation.ConclusionIncreased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.

Molecular docking studies of ephedrine, eugenol, and their derivatives as arginase inhibitors: Implications in asthma

Background: Arginine being a common substrate for arginase and nitric oxide synthase (NOS) an imbalance between enzymes could lead to a shift in airway responses. Reports suggest that increased arginase reduces the substrate availability to NOS and attributes to the airway hyperresponsiveness. Hence, inhibition of arginase might enhance the bioavailability of arginine to NOS and generates nitric oxide (NO) a bronchodilator. Molecules from Ephedra and Eugenia caryophyllus are documented for bronchodilator properties. However, the mechanism of action of these molecules for enhancing bronchodilation is not well characterized. The objective of the present study is to assess whether these molecules could inhibit the arginase by binding at its active site and helps in bronchodilation using in silico approach. Methods: The crystal structures of the arginase and NOS enzymes were selected from the protein database. The molecules from Ephedra and Eugenia caryophyllus were obtained from Pubchem. Drug likeliness and bioactivity of the molecules were assessed by Molinspiration. The successful molecules were docked with active sites of enzymes using docking software, and the docked complexes were analyzed using Accelrys Discovery Studio. Results: Molecules from Ephedra and Eugenia caryophyllus were able to interact to arginase at the active site whereas away from the active site in case of NOS. The molecules showed differential binding affinities, and some of them had higher binding affinity than substrate arginine. Conclusion: In silico study suggests that molecules of Ephedra and Eugenia were capable of blocking the active site of arginase. We speculate that if these molecules are used as therapeutics, they could inhibit the arginase activity and this might increase arginine availability to NOS to produce NO which acts as bronchodilator. Our study suggests that molecules which bind to active site of arginine and do not affect the active site of NOS might be the potential molecules for arginase associated asthma.

6-Gingerol as an arginase inhibitor prevents urethane-induced lung carcinogenesis by reprogramming tumor supporting M2 macrophages to M1 phenotype.

6-Gingerol (6-G) is the main bioactive component in Ginger (Zingiber officinale Roscoe). The aim of this study was to explore the contribution of macrophage polarization in 6-G-associated anti-cancer effects. In a urethane-induced lung carcinogenic model, lung carcinogenesis was positively correlated with macrophage (F4/80+) infiltration in lung interstitial in the control group. Furthermore, higher numbers of arginase+/F4/80+ M2 cells than iNOS+/F4/80+ M1 cells were observed in interstitial macrophages. Moreover, macrophage depletion by liposome-encapsulated clodronate (LEC) could significantly prevent lung carcinogenesis, whereas pexidartinib promoted lung carcinogenesis. After 6-G treatment, lung carcinogenesis was ameliorated with increased M1 macrophages and decreased M2 macrophages in the lung interstitial. ELISA showed that the levels of IFN-γ and IL-12 increased and the levels of IL-10 and TGF-β1 decreased in the alveolar cavity compared to those in the control group. Unexpectedly, the carcinogenesis-preventing efficacy of 6-G was promoted in LEC-treated mice, but completely aborted in pexidartinib-treated mice. In the in vitro experiment, 6-G reset the IL-4-induced arginase+ M2 cells toward iNOS+ M1 cells and exhibited reduced levels of arginase 1 and ROS and elevated levels of L-arginine and NO. LEC and nor-NOHA selectively suppressed M2 macrophages but had a negligible effect on M1 macrophages, whereas pexidartinib decreased both M2 and M1 macrophages. The iNOS+ macrophage-promoting efficacy of 6-G was increased by LEC, but was completely eliminated by pretreatment with pexidartinib or nor-NOHA. M2 macrophage-resetting efficacy of 6-G was confirmed in a Lewis lung cancer allograft model. This study indicated a reprogramming effect of 6-G as an arginase inhibitor on tumor supporting macrophages.