Arginase Inhibitor Research Articles

EP258: Pre-analytical considerations are important in the pharmacological assessment of enzyme therapeutics

Enzyme therapeutics are distinct from other modalities as they have the potential to continue metabolizing their target analyte even after blood collection. This poorly characterized phenomenon is important as it may lead to an overestimation of the in vivo pharmacological effects of an enzyme therapeutic. With the recent development of highly effective metabolite lowering enzyme therapeutics, it is important to understand the impact of pre-analytical sample processing as a source of inaccurate metabolite measurements in pharmacological evaluation of both nonclinical and clinical studies. Pegzilarginase is a pegylated modified human arginase 1 enzyme in clinical development for Arginase 1 deficiency (ARG1-D). Given the potency of pegzilarginase, the potential for spurious measurements of plasma arginine levels was investigated. Continued metabolism of arginine was demonstrated ex vivo after blood draw leading to artefactually low arginine levels in normally processed plasma samples. These effects were ameliorated by the competitive arginase inhibitor nor-NOHA when added to the collection tube prior to blood collection. This approach has been adapted to ensure accurate measurement of plasma arginine in patients receiving pegzilarginase in clinical trials. Similarly, a recently developed engineered enzyme (AGLE-177), with novel specificity for homocysteine and homocystine has been shown to artefactually lower total homocysteine plasma levels ex vivo after blood collection. Mitigation of the effect, by adding an inhibitor to the blood collection tube, enabled accurate assessment of tHcy levels in non-clinical efficacy and toxicology studies. These studies highlight the need to consider continued enzymatic digestion of metabolites during the pre-analytical sample handling for in vivo pharmacological studies of enzyme therapeutics.

Effects of Chronic Arginase Inhibition with Norvaline on Tau Pathology and Brain Glucose Metabolism in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is an insidious neurodegenerative disorder representing a serious continuously escalating medico-social problem. The AD-associated progressive dementia is followed by gradual formation of amyloid plaques and neurofibrillary tangles in the brain. Though, converging evidence indicates apparent metabolic dysfunctions as key AD characteristic. In particular, late-onset AD possesses a clear metabolic signature. Considerable brain insulin signaling impairment and a decline in glucose metabolism are common AD attributes. Thus, positron emission tomography (PET) with glucose tracers is a reliable non-invasive tool for early AD diagnosis and treatment efficacy monitoring. Various approaches and agents have been trialed to modulate insulin signaling. Accumulating data point to arginase inhibition as a promising direction to treat AD via diverse molecular mechanisms involving, inter alia, the insulin pathway. Here, we use a transgenic AD mouse model, demonstrating age-dependent brain insulin signaling abnormalities, reduced brain insulin receptor levels, and substantial energy metabolism alterations, to evaluate the effects of arginase inhibition with Norvaline on glucose metabolism. We utilize fluorodeoxyglucose whole-body micro-PET to reveal a significant treatment-associated increase in glucose uptake by the brain tissue in-vivo. Additionally, we apply advanced molecular biology and bioinformatics methods to explore the mechanisms underlying the effects of Norvaline on glucose metabolism. We demonstrate that treatment-associated improvement in glucose utilization is followed by significantly elevated levels of insulin receptor and glucose transporter-3 expression in the mice hippocampi. Additionally, Norvaline diminishes the rate of Tau protein phosphorylation. Our results suggest that Norvaline interferes with AD pathogenesis. These findings open new avenues for clinical evaluation and innovative drug development.

Alterations of nitric oxide homeostasis as trigger of intestinal barrier dysfunction in non-alcoholic fatty liver disease.

Changes in intestinal nitric oxide metabolism are discussed to contribute for the development of intestinal barrier dysfunction in non‐alcoholic fatty liver disease (NAFLD). To induce steatosis, female C57BL/6J mice were pair‐fed with a liquid control diet (C) or a fat‐, fructose‐ and cholesterol‐rich diet (FFC) for 8 weeks. Mice received the diets ± 2.49 g L‐arginine/kg bw/day for additional 5 weeks. Furthermore, mice fed C or FFC ± L‐arginine/kg bw/day for 8 weeks were concomitantly treated with the arginase inhibitor Nω‐hydroxy‐nor‐L‐arginine (nor‐NOHA, 0.01 g/kg bw). Liver damage, intestinal barrier function, nitric oxide levels and arginase activity in small intestine were assessed. Also, arginase activity was measured in serum from 13 patients with steatosis (NAFL) and 14 controls. The development of steatosis with beginning inflammation was associated with impaired intestinal barrier function, increased nitric oxide levels and a loss of arginase activity in small intestine in mice. L‐arginine supplementation abolished the latter along with an improvement of intestinal barrier dysfunction; nor‐NOHA attenuated these effects. In patients with NAFL, arginase activity in serum was significantly lower than in healthy controls. Our data suggest that increased formation of nitric oxide and a loss of intestinal arginase activity is critical in NAFLD‐associated intestinal barrier dysfunction.

In silico design and in vitro assessment of anti-Helicobacter pylori compounds as potential small-molecule arginase inhibitors.

Related to a variety of gastrointestinal disorders ranging from gastric ulcer to gastric adenocarcinoma, the infection caused by the gram-negative bacteria Helicobacter pylori (H. pylori) poses as a great threat to human health; hence, the search for new treatments is a global priority. The H. pylori arginase (HPA) protein has been widely studied as one of the main virulence factors of this bacterium, being involved in the prevention of nitric oxide-mediated bacterial cell death, which is a central component of innate immunity. Given the growing need for the development of new drugs capable of combating the infection by H. pylori, the present work describes the search for new HPA inhibitors, using virtual screening techniques based on molecular docking followed by the evaluation of the proposed modes of interaction at the HPA active site. In vitro studies of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), followed by cytotoxicity activity in gastric adenocarcinoma and non-cancer cells, were performed. The results highlighted compounds 6, 11, and 13 as potential inhibitors of HPA; within these compounds, the results indicated 13 presented an improved activity toward H. pylori killing, with MIC and MBC both at 64µg/mL. Moreover, compound 13 also presented a selectivity index of 8.3, thus being more selective for gastric adenocarcinoma cells compared to the commercial drug cisplatin. Overall, the present work demonstrates the search strategy based on in silico and in vitro techniques is able to support the rational design of new anti-H. pylori drugs.

The effect of coal-derived humic substances and their silver-containing bionanocomposites on arginine balance in peritoneal macrophages of intact mice

Background. Antigen-presenting cells (APCs), especially macrophages, play an important role in the body defense against various pathogens. Their dysfunction and polarization are associated with most inflammatory and autoimmune diseases. The inflammatory process is regulated by activation and / or inhibition of genes differentially expressed by macrophages. Successful correction of inflammation leads firstly to elimination of inflammatory stimuli and then to remodeling and restoration of tissues and organs. It was experimentally confirmed that silvercontaining bionanocomposites based on natural humic substances (HS) obtained from coal of different origin, as well as initial matrices of these HS, are capable of activating pro- and anti-inflammatory properties of macrophages.Aim. To study cytotoxic, pyrogenic, and immunomodulatory properties (arginine balance) of initial HS samples and samples of silver nanoparticles ultradispersed in these HS matrices (HS-AgNPs) in the cell culture of peritoneal macrophages, as well as their effect on pro- and anti-inflammatory properties of APCs.Materials and methods. Cultural and biochemical methods were used in the study.Results. The study showed that the samples CHE-K, CHE-AgNPs, CHS-K, and CHP-K increased M1 macrophage polarization due to stimulation of the NO-synthase activity and inhibition of arginase. The samples CHI-K, CHIAgNPs, CHP-AgNPs, and CHS-AgNPs modulated an alternative M2 or M2-like state of macrophage activation. At the same time, HS are not cytotoxic at effective concentrations, and three out of four studied samples did not contain pyrogenic impurities.Conclusion. The use of HS and their silver-containing bionanocomposites, which have the ability to greatly affect the polarization of antigen-presenting cells, is a promising research area in correction of the inflammatory response for solving an important social and medical problem of treating chronic wounds.

Evidence of Suppression of Tumor Growth by Extract of Rumex Obtusifolius in Combination with Inhibitors of Arginase and Nitric Oxide Synthase: Role of Oxidative Stress, Inflammation, and Polyamine Synthesis

Evidence of Suppression of Tumor Growth by Extract of Rumex Obtusifolius in Combination with Inhibitors of Arginase and Nitric Oxide Synthase: Role of Oxidative Stress, Inflammation, and Polyamine Synthesis

ОБ УЧАСТИИ АРГИНАЗЫ ПЕЧЕНИ В ПРОЦЕССАХ ДЕТОКСИКАЦИИ И РАЗВИТИЯ ОКСИДАТИВНОГО СТРЕССА У КРЫС В УСЛОВИЯХ АЛКОГОЛЬНОЙ ИНТОКСИКАЦИИ РАЗЛИЧНОЙ ТЯЖЕСТИ

Modern medicine faces the problem of the steady growth of alcoholic pathology. And as you know, morbidity and mortality with regular consumption of alcoholic beverages is associated with the toxic effects of ethanol on the most important human organs and, first of all, the liver. To date, a sufficient number of facts have accumulated indicating the importance of liver arginase in vital processes in health and disease. The aim of the study was to elucidate the significance of liver arginase in the detoxification processes and the development of oxidative stress in rats with chronic ethanol intoxication of different severity. In experiments on rats using modern physiological, biochemical research methods and a pharmacological approach, it was found that liver arginase participate in changes in liver detoxification function and the development of oxidative stress induced by chronic ethanol intoxication. The direction and severity of changes in arginase activity and liver detoxification function during chronic alcoholism depends on the severity of chronic alcohol intoxication. Under the influence of daily intragastric administration for 60 days, a 30% aqueous solution of ethanol (3.5 g 92% ethanol per kg of body weight) in animals under conditions of development of oxidative stress inhibited the activity of arginase and detoxification function of the liver, and the introduction of 10% aqueous solution of ethanol (1.0 g 92% ethanol per kg of body weight) for 2 months leads to an increase in the activity of liver arginase and detoxification processes. The action in the body of the arginase inhibitor Nω-hydroxy-nor-L-arginine contributes to the development of characteristic changes in the processes of detoxification and lipid peroxidation in the liver during chronic alcohol intoxication caused by intragastric the introduction of ethanol at a dose of 3.5 g / kg for 60 days.

Benzimidazole Derivatives as New and Selective Inhibitors of Arginase from Leishmania mexicana with Biological Activity against Promastigotes and Amastigotes.

Leishmaniasis is a disease caused by parasites of the Leishmania genus that affects 98 countries worldwide, 2 million of new cases occur each year and more than 350 million people are at risk. The use of the actual treatments is limited due to toxicity concerns and the apparition of resistance strains. Therefore, there is an urgent necessity to find new drugs for the treatment of this disease. In this context, enzymes from the polyamine biosynthesis pathway, such as arginase, have been considered a good target. In the present work, a chemical library of benzimidazole derivatives was studied performing computational, enzyme kinetics, biological activity, and cytotoxic effect characterization, as well as in silico ADME-Tox predictions, to find new inhibitors for arginase from Leishmania mexicana (LmARG). The results show that the two most potent inhibitors (compounds 1 and 2) have an I50 values of 52 μM and 82 μM, respectively. Moreover, assays with human arginase 1 (HsARG) show that both compounds are selective for LmARG. According to molecular dynamics simulation studies these inhibitors interact with important residues for enzyme catalysis. Biological activity assays demonstrate that both compounds have activity against promastigote and amastigote, and low cytotoxic effect in murine macrophages. Finally, in silico prediction of their ADME-Tox properties suggest that these inhibitors support the characteristics to be considered drug candidates. Altogether, the results reported in our study suggest that the benzimidazole derivatives are an excellent starting point for design new drugs against leishmanisis.

Amelioration of the Abnormalities Associated with Metabolic Syndrome by L-Norvaline in Hyperlipidemic Diabetic Rats

Abstract The present study was designed to assess the treatment effect of arginase inhibitor, L-Norvaline in abnormalities associated with high fat diet (HFD) and fructose-induced metabolic syndrome. The HFD and fructose was fed to the rats for a period of 45 days. Animals having body weight of 350 g and fasting blood sugar level of more than 250 mg/dl were considered as hyperlipidemic diabetic rats (HDR) and selected for the study. The HDR were divided into three groups having six animals each. The HDR received L–Norvaline (10 mg/kg/day, i.p.) and standard drug, gemfibrozil (60 mg/kg/day, p.o.), for a period of 30 days. Various hormonal, biochemical and tissue parameters were evaluated at the end of the study. Both treatments significantly decreased body weight, BMI, fasting blood sugar and insulin level and improved insulin resistance in HDR as compared to the toxicant control group. A significant improvement was observed in the lipid profile, levels of nitrate, leptin, C-reactive protein and adiponectin in HDR. L-Norvaline also caused slight decrease in the malondialdehyde level, though, no prominent effect was observed on the level of superoxide dismutase and reduced glutathione in the pancreas of HDR, as compared to the toxicant control group. L-Norvaline treatment also improved the histo-architecture of pancreatic cells. Results of the present study concludes that L-Norvaline caused significant alleviation of the abnormalities of MetS indicating that it can be used as potential treatment strategy for managing the symptoms of metabolic syndrome.

Increased Lung Uric Acid Deteriorates Pulmonary Arterial Hypertension.

BackgroundRecent studies have demonstrated that uric acid (UA) enhances arginase activity, resulting in decreased NO in endothelial cells. However, the role of lung UA in pulmonary arterial hypertension (PAH) remains uncertain. We hypothesized that increased lung UA level contributes to the progression of PAH.Methods and ResultsIn cultured human pulmonary arterial endothelial cells, voltage‐driven urate transporter 1 (URATv1) gene expression was detected, and treatment with UA increased arginase activity. In perfused lung preparations of VEGF receptor blocker (SU5416)/hypoxia/normoxia‐induced PAH model rats, addition of UA induced a greater pressure response than that seen in the control and decreased lung cGMP level. UA‐induced pressor responses were abolished by benzbromarone, a UA transporter inhibitor, or L‐norvaline, an arginase inhibitor. In PAH model rats, induction of hyperuricemia by administering 2% oxonic acid significantly increased lung UA level and induced greater elevation of right ventricular systolic pressure with exacerbation of occlusive neointimal lesions in small pulmonary arteries, compared with nonhyperuricemic PAH rats. Administration of benzbromarone to hyperuricemic PAH rats significantly reduced lung UA levels without changing XOR (xanthine oxidoreductase) activity, and attenuated right ventricular systolic pressure increase and occlusive lesion development. Topiroxostat, a XOR inhibitor, significantly reduced lung XOR activity in PAH rats, with no effects on increase in right ventricular systolic pressure, arterial elastance, and occlusive lesions. XOR‐knockout had no effects on right ventricular systolic pressure increase and arteriolar muscularization in hypoxia‐exposed mice.ConclusionsIncreased lung UA per se deteriorated PAH, whereas XOR had little impact. The mechanism of increased lung UA may be a novel therapeutic target for PAH complicated with hyperuricemia.

Abstract PO-116: Deletion of Arginase 1 in myeloid cells alters the pancreatic cancer microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDA) is a deadly disease with a 5-year survival of only 10%. PDA is characterized by an abundant fibroinflammatory stroma that includes abundant fibroblasts and immune cells, mainly myeloid cells. Infiltrating myeloid cells express high levels of Arginase 1 (Arg1), an enzyme that metabolizes L-arginine. Conversely, CD8+ T cells are scarce in PDA, and when present have an overwhelmingly exhausted phenotype. Whether myeloid cell Arginase is a key driver of immune suppression in pancreatic cancer is unknown. Here, we tested the hypothesis that myeloid cells in the tumor microenvironment mediate immune suppression in PDA through expression of Arg1. To test this hypothesis, we used a combination of genetically engineered pancreatic cancer mouse models and pharmacological approaches. Using a FlpO- and Cre-based dual recombinase system, we have generated a mouse model that develops pancreatic cancer spontaneously because of oncogenic Kras expression in the epithelium, while at the same time lacking Arg1 expression in the myeloid cell compartment (Ptf1a-FlpO/+;KrasFrt-STOP-Frt-G12D/+;LysMCre;Arg1f/f). To complement the genetic model and inhibit the function of Arginase systemically, we used an Arginase inhibitor (CB-1158, Incyte, Inc.) alone and in combination with an immune checkpoint blockade (anti-PD1). Using these multiple approaches, we observed decrease progression to invasive disease in the genetic model, and sensitization to immune checkpoint treatment in the transplantation model. In both settings, changes in tumor growth were accompanied by an increase in CD8+ T cell infiltration and activation. These changes support the notion that myeloid Arg1 is mediator of immune suppression in PDA, and a potential therapeutic target. Citation Format: Rosa E. Menjivar, Zeribe Nwosu, Wenting Du, Katelyn Donahue, Carlos Espinoza, Ashley Velez-Delgado, Kristee Brown, Wei Yan, Christopher Halbrook, Yaqing Zhang, Costas Lyssiotis, Marina Pasca di Magliano. Deletion of Arginase 1 in myeloid cells alters the pancreatic cancer microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-116.

Biliary Tract Cancers: Treatment Updates and Future Directions in the Era of Precision Medicine and Immuno-Oncology.

The effective management of biliary tract cancers (BTCs) has been hampered by limited options for systemic therapy. In recent years, the focus on precision medicine has made technologies such as next-generation sequencing (NGS) accessible to clinicians to identify targetable mutations in BTCs in tumor tissue (primarily) as well as blood, and to treat them with targeted therapies when possible. It has also expanded our understanding of functional pathways associated with genetic alterations and opened doors for identifying novel targets for treatment. Recent advances in the precision medicine approach allowed us to identify new molecular markers in BTCs, such as epigenetic changes (methylation and histone modification) and non-DNA markers such as messenger RNA, microRNA, and long non-coding RNA. It also made detecting these markers from non-traditional sources such as blood, urine, bile, and cytology (from fine-needle aspiration and biliary brushings) possible. As these tests become more accessible, we can see the integration of different molecular markers from all available sources to aid physicians in diagnosing, assessing prognosis, predicting tumor response, and screening BTCs. Currently, there are a handful of approved targeted therapies and only one class of immunotherapy agents (immune checkpoint inhibitors or ICIs) to treat BTCs. Early success with new targets, vascular endothelial growth factor receptor (VEGFR), HER2, protein kinase receptor, and Dickkopf-1 (DKK1); new drugs for known targets, fibroblast growth factor receptors (FGFRs) such as futabatinib, derazantinib, and erdafitinib; and ICIs such as durvalumab and tremelimumab is encouraging. Novel immunotherapy agents such as bispecific antibodies (bintrafusp alfa), arginase inhibitors, vaccines, and cellular therapy (chimeric antigen receptor—T cell or CAR-T, natural killer cells, tumor-infiltrating lymphocytes) have the potential to improve outcomes of BTCs in the coming years.

TGF-β orchestrates the phenotype and function of monocytic myeloid-derived suppressor cells in colorectal cancer

BackgroundMonocytic myeloid-derived suppressor cells (M-MDSCs) are significantly expanded in the blood of colorectal cancer (CRC) patients. However, their presence and underlying mechanisms in the tumour microenvironment of CRC have not been examined in detail.MethodsTumour tissues and peripheral blood from CRC patients were analysed for the presence of M-MDSCs. The mechanisms of suppression were analysed by blocking pathways by which MDSCs abrogate T cell proliferation. Co-culture of CRC cells with monocytes were performed with and without cytokine blocking antibodies to determine the mechanism by which CRC cells polarise monocytes. Multi-spectral IHC was used to demonstrate the intra-tumoral location of M-MDSCs.ResultsTumour tissues and blood of CRC patients contain M-MDSCs which inhibit T cell proliferation. Whilst inhibition of arginase and nitric oxide synthase 2 fail to rescue T cell proliferation, blockade of IL-10 released by these HLA-DR− cells abrogates the suppresivity of M-MDSCs. Tumour conditioned media (TCM) significantly reduces HLA-DR expression, increases IL-10 release from monocytes and causes them to become suppressive. TGF-β is highly expressed in the TCM and accumulates in the plasma. TGF-β reduces HLA-DR expression and drives monocyte immunosuppressivity. The invasive margin of CRC is enriched in CD14+ HLA-DR− cells in close proximity to T cells.ConclusionsOur study demonstrates the cross-talk between CRC cells, M-MDSCs and T cells. Characterisation of CRC M-MDSCs point to therapeutic avenues to target these cells in addition to TGF-β blockade.

Arginase inhibition by rhaponticin increases L-arginine concentration that contributes to Ca2+-dependent eNOS activation

Although arginase primarily participates in the last reaction of the urea cycle, we have previously demonstrated that arginase II is an important cytosolic calcium regulator through spermine production in a p32-dependent manner. Here, we demonstrated that rhaponticin (RPT) is a novel medicinal-plant arginase inhibitor and investigated its mechanism of action on Ca2+-dependent endothelial nitric oxide synthase (eNOS) activation. RPT was uncompetitively inhibited for both arginases I and II prepared from mouse liver and kidney. It also inhibited arginase activity in both aorta and human umbilical vein endothelial cells (HUVECs). Using both microscope and FACS analyses, RPT treatments induced increases in cytosolic Ca2+ levels using Fluo-4 AM as a calcium indicator. Increased cytosolic Ca2+ elicited the phosphorylations of both CaMKII and eNOS Ser1177 in a time-dependent manner. RPT incubations also increased intracellular L-arginine (L-Arg) levels and activated the CaMKII/AMPK/Akt/eNOS signaling cascade in HUVECs. Treatment of L-Arg and ABH, arginase inhibitor, increased intracellular Ca2+ concentrations and activated CaMKII-dependent eNOS activation in ECs of WT mice, but, the effects were not observed in ECs of inositol triphosphate receptor type 1 knockout (IP3R1−/−) mice. In the aortic endothelium of WT mice, RPT also augmented nitric oxide (NO) production and attenuated reactive oxygen species (ROS) generation. In a vascular tension assay using RPT-treated aortic tissue, cumulative vasorelaxant responses to acetylcholine (Ach) were enhanced, and phenylephrine (PE)-dependent vasoconstrictive responses were retarded, although sodium nitroprusside and KCl responses were not different. In this study, we present a novel mechanism for RPT, as an arginase inhibitor, to increase cytosolic Ca2+ concentration in a L-Arg-dependent manner and enhance endothelial function through eNOS activation.

An Epidermal-Specific Role for Arginase1 during Cutaneous Wound Repair

Nonhealing wounds are a major area of unmet clinical need remaining problematic to treat. Improved understanding of prohealing mechanisms is invaluable. The enzyme arginase1 (ARG1) is involved in prohealing responses, with its role in macrophages best characterized. ARG1 is also expressed by keratinocytes; however, ARG1 function in these critical wound repair cells is not understood. We characterized ARG1 expression in keratinocytes during normal cutaneous repair and reveal de novo temporal and spatial expression at the epidermal wound edge. Interestingly, epidermal ARG1 expression was decreased in both human and murine delayed healing wounds. We therefore generated a keratinocyte-specific ARG1-null mouse model (K14-cre;Arg1fl/fl) to explore arginase function. Wound repair, linked to changes in keratinocyte proliferation, migration, and differentiation, was significantly delayed in K14-cre;Arg1fl/fl mice. Similarly, using the arginase inhibitor N(omega)-hydroxy-nor-L-arginine, human in vitro and ex vivo models further confirmed this finding, revealing the importance of the downstream polyamine pathway in repair. Indeed, restoring the balance in ARG1 activity through the addition of putrescine proved beneficial in wound closure. In summary, we show that epidermal ARG1 plays, to our knowledge, a previously unreported intrinsic role in cutaneous healing, highlighting epidermal ARG1 and the downstream mediators as potential targets for the therapeutic modulation of wound repair.

529: A phase 1b, randomized, double-blind, placebo-controlled, dose-escalation trial of CB-280, an arginase inhibitor, in patients with cystic fibrosis

529: A phase 1b, randomized, double-blind, placebo-controlled, dose-escalation trial of CB-280, an arginase inhibitor, in patients with cystic fibrosis

Comprehensive Strategies to Bicyclic Prolines: Applications in the Synthesis of Potent Arginase Inhibitors.

Comprehensive synthetic strategies afforded a diverse set of structurally unique bicyclic proline-containing arginase inhibitors with a high degree of three-dimensionality. The analogs that favored the Cγ-exo conformation of the proline improved the arginase potency over the initial lead. The novel synthetic strategies reported here not only enable access to previously unknown stereochemically complex proline derivatives but also provide a foundation for the future synthesis of bicyclic proline analogs, which incorporate inherent three-dimensional character into building blocks, medicine, and catalysts and could have a profound impact on the conformation of proline-containing peptides and macrocycles.

Hepatic Ischemia-Reperfusion Impairs Blood-Brain Barrier Partly Due to Release of Arginase From Injured Liver.

Aim: Hepatic ischemia-reperfusion (HIR) induces remote organs injury, including the brain. The homeostasis of the brain is maintained by the blood-brain barrier (BBB); thus, we aimed to investigate whether HIR impaired BBB and attempted to elucidate its underlying mechanism. Methods: Cell viability of human cerebral microvascular endothelial cells (hCMEC/D3) was measured following 24 h incubation with a serum of HIR rat undergoing 1 h ischemia and 4 h reperfusion, liver homogenate, or lysate of primary hepatocytes of the rat. The liver homogenate was precipitated using (NH4)2SO4 followed by separation on three columns and electrophoresis to identify the toxic molecule. Cell activity, apoptosis, proliferation, cell cycle, and expressions of proteins related to cell cycle were measured in hCMEC/D3 cells incubated with identified toxic molecules. HIR rats undergoing 1 h ischemia and 24 h reperfusion were developed to determine the release of an identified toxic molecule. BBB function was indexed as permeability to fluorescein and brain water. Endothelial cell proliferation and expressions of proteins related to the cell cycle in cerebral microvessels were measured by immunofluorescence and western blot. Results: Toxic molecule to BBB in the liver was identified to be arginase. Arginase inhibitor nor-NOHA efficiently attenuated hCMEC/D3 damage caused by liver homogenate and serum of HIR rats. Both arginase and serum of HIR rats significantly lowered arginine (Arg) in the culture medium. Arg addition efficiently attenuated the impairment of hCMEC/D3 caused by arginase or Arg deficiency, demonstrating that arginase impaired hCMEC/D3 via depriving Arg. Both arginase and Arg deficiency damaged hCMEC/D3 cells by inhibiting cell proliferation, retarding the cell cycle to G1 phase, and downregulating expressions of cyclin A, cyclin D, CDK2, and CDK4. HIR notably increased plasma arginase activity and lowered Arg level, increased the BBB permeability accompanied with enhanced brain water, and decreased the proliferative cells (marked by Ki67) in cerebral microvessels (marked by CD31) and protein expressions of cyclin A, cyclin D, CDK2 and CDK4 in isolated brain microvessels. Oral supplement of Arg remarkably attenuated these HIR-induced alterations. Conclusion: HIR leads to substantial release of arginase from the injured liver and then deprives systemic Arg. The Arg deficiency further impairs BBB via inhibiting the proliferation of brain microvascular endothelial cells by cell cycle arrest.

Erythrocytes induce vascular dysfunction in COVID-19

BackgroundVascular injury has been implicated as a major cause of clinical complications in patients with coronavirus disease 2019 (COVID-19). Autopsy studies have revealed destruction of the endothelial cell lining, which might explain cardiovascular alterations arising from the infection. However, data demonstrating endothelial dysfunction during ongoing infection are sparse, and the underlying mechanisms are still largely unknown. Red blood cells (RBCs) are affected by COVID-19 with alterations in their structure and function, possibly contributing to vascular injury via increased oxidative stress.PurposeTo determine the presence of endothelial dysfunction in patients with COVID-19 and to explore the RBC as a possible mediator of such dysfunction.MethodsThe study was performed on 17 patients hospitalized for moderate COVID-19 infection and age- and sex-matched healthy subjects. Inclusion criteria of the COVID-19 patients were PCR-verified SARS-CoV2 infection, pulmonary infiltrates on x-ray, oxygen demand during hospital stay and ≤ one cardiovascular co-morbidity. Microvascular endothelial function in vivo was assessed with a pulse amplitude tonometry device on each index finger at baseline and during reactive hyperemia and expressed as reactive hyperemia index (RHI). RBCs from COVID-19 patients (C19-RBCs) and healthy subjects (H-RBCs) were incubated with isolated rat aortic segments for evaluation of endothelium-dependent and -independent relaxation.ResultsCOVID-19 patients displayed profound impairment in endothelial function in vivo with RHI 1.56 (1.30–1.81, median and interquartile range) compared to healthy subjects 2.36 (1.97–2.79, p<0.001). C19-RBCs induced severe impairment in both endothelium-dependent (27% maximal relaxation) and -independent relaxations (54%) compared to H-RBCs (67% and 95% relaxation, respectively). Further, C19-RBCs induced upregulation of vascular arginase 1 (∼2 fold increase compared to H-RBCs) and markers of oxidative stress (∼6 fold). Consequently, inhibition of vascular arginase or superoxide attenuated the impairment in endothelial function induced by C19-RBCs. C19-RBCs were characterized by increased production of reactive oxygen species (∼1.4 fold) and reduced export of the nitric oxide metabolite nitrate. Following pre-incubation with interferon-γ, but not interleukin-6 or tumor necrosis factor-α, H-RBCs induced impairment in endothelial function.ConclusionsThis study demonstrates the presence of marked endothelial dysfunction in an otherwise mainly healthy patient group hospitalized for COVID-19, and clearly implicates a central role of the RBC as a mediator of endothelial injury through enhancement of reactive oxygen species and arginase. These data shed light on a new pathological mechanism underlying vascular dysfunction in COVID-19 patients and may lay the foundation for future therapeutic developments.Funding AcknowledgementType of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung foundationSwedish Research Council

Temporal relationship between systemic endothelial dysfunction and alterations in erythrocyte function in a murine model of chronic heart failure.

AimsEndothelial dysfunction (ED) and red blood cell distribution width (RDW) are both prognostic factors in heart failure (HF), but the relationship between them is not clear. In this study, we used a unique mouse model of chronic HF driven by cardiomyocyte-specific overexpression of activated Gαq protein (Tgαq*44 mice) to characterize the relationship between the development of peripheral ED and the occurrence of structural nanomechanical and biochemical changes in red blood cells (RBCs).Methods and resultsSystemic ED was detected in vivo in 8-month-old Tgαq*44 mice, as evidenced by impaired acetylcholine-induced vasodilation in the aorta and increased endothelial permeability in the brachiocephalic artery. ED in the aorta was associated with impaired nitric oxide (NO) production in the aorta and diminished systemic NO bioavailability. ED in the aorta was also characterized by increased superoxide and eicosanoid production. In 4- to 6-month-old Tgαq*44 mice, RBC size and membrane composition displayed alterations that did not result in significant changes in their nanomechanical and functional properties. However, 8-month-old Tgαq*44 mice presented greatly accentuated structural and size changes and increased RBC stiffness. In 12-month-old Tgαq*44 mice, the erythropathy was featured by severely altered RBC shape and elasticity, increased RDW, impaired RBC deformability, and increased oxidative stress (gluthatione (GSH)/glutathione disulfide (GSSG) ratio). Moreover, RBCs taken from 12-month-old Tgαq*44 mice, but not from 12-month-old FVB mice, coincubated with aortic rings from FVB mice, induced impaired endothelium-dependent vasodilation and this effect was partially reversed by an arginase inhibitor [2(S)-amino-6-boronohexanoic acid].ConclusionIn the Tgαq*44 murine model of HF, systemic ED accelerates erythropathy and, conversely, erythropathy may contribute to ED. These results suggest that erythropathy may be regarded as a marker and a mediator of systemic ED in HF. RBC arginase and possibly other RBC-mediated mechanisms may represent novel therapeutic targets for systemic ED in HF.